Substituted esters containing polyols and saccharides for treating hepatotoxicity and fatty liver diseases

ABSTRACT

The present invention relates to compounds effective in treating hepatotoxicity and fatty liver diseases and uses thereof. The present compound is represented by Formula (II), which has the formula: R 1 —O—X—(CH 2 ) m —X—O—R 2 , wherein: each X is —C(═O)—; R 1  is a C 1 -C 18  alkyl polyol: R 2  is a saccharide group of formula (G) p ; G is a monosaccharide residue, where (i) at least one of the —OH groups in (G) p  is substituted by a halogen atom, and (ii) the saccharide group of formula (G) p  is linked to —O— through a —CH 2  group; p is 1 or 2; and m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

RELATED APPLICATION

This application is a Divisional of U.S. application Ser. No.15/564,526, filed Oct. 5, 2017, which is the National Phase of PCTInternational Application No. PCT/CN2016/100187, filed on Sep. 26, 2016,which claims priority under 35 U.S.C. 119(e) to U.S. ProvisionalApplication No. 62/222,959, filed on Sep. 24, 2015 and 62/257,697, filedon Nov. 19, 2015 and under 35 U.S.C. 119(a) to Patent Application No.PCT/CN2016/078039, filed in China on Mar. 31, 2016, all of which arehereby expressly incorporated by reference into the present application.

TECHNOLOGY FIELD

The present invention relates to compounds effective in treatinghepatotoxicity and fatty liver diseases and uses thereof.

BACKGROUND OF THE INVENTION

Injuries in organs may be caused by toxic agents such as a therapeuticdrug when administered overdose which often leads to injuries in organsespecially liver or kidney. Acetaminophen (also known as Panadol) isalso called paracetamol or N-acetyl-para-aminophenol (APAP) and is themost widely used pain-relieving and fever-reducing drug on the market.Each year, numerous cases of drug intoxication or suicide are reporteddue to improper use of APAP, and liver damage caused by APAP is the maincause of severe diseases and death. Alcohols or organic solvents such ascarbon tetrachloride (CCl₄) may also cause hepatotoxicity. A number ofclinical studies have demonstrated that hepatotoxicity induced by APAPis preventable and early diagnosis along with real-time administrationof the antidote N-acetylcysteine (NAC) can prevent the occurrence ofhepatotoxicity.

Early detection of acetaminophen overdose is necessary because the bestprognosis can be achieved if the antidote is given within 8 hours afterpoisoning. The early signs of drug intoxication include discomfort,nausea and vomiting. However, some patients may show no signs ofintoxication at the early stage (stage 1) even if their bloodconcentrations of acetaminophen are at the poisoning levels and theirabnormal liver function is apparently abnormal. The signs ofhepatotoxicity, such as abdominal pain, persistent vomiting, jaundice,right upper quadrant pain, usually become apparent 24-48 hours afteringestion of a significant amount of acetaminophen (stage 2). Serumamintransferase usually starts to rise 16 hours after administrationwith clinical symptoms. Stage 3 usually occurs 3-4 days afteradministration and the degree of liver damage as well as prognosis canbe well predicted at the time. The signs of hepatotoxicity progress frommild symptoms with elevated liver function values (AST>1,000 IU/L) tosevere acute fulminant hepatitis accompanied by metabolic acidosis,jaundice, hyperglycemia, AST>1,000 IU/L, abnormal blood clotting andhepatic/brain lesions. Stage 4 will cause oliguria renal failure ordeath in severe cases.

Some patients with acetaminophen intoxication show only mild liverdamage but with severe renal toxicity which is mainly caused by directmetabolism of APAP in P-450s (cytochrome P450s⋅CYPs) of the renaltubule. Nonetheless, acute renal failure may also result fromhepatorenal syndrome caused by acute liver failure and the fractionexcretion of Na (FeNa) can be used for differentiation primary renaldamage (FeNa>1) from hepatorenal syndrome (FeNa>1). The calculationformula for FeNa is (Sodium urinary÷Creatinine urinary)÷(Sodiumplasma÷Creatinine plasma)×100.

The peak concentration of acetaminophen in blood is achieved 1-2 hoursafter oral administration and a significant amount is eliminated byliver, more than 90% is conjugated to glucuronide and sulfate and formnon-toxic metabolites and only less than 5% is eliminated by differentCYPs, including CYP2E1, CYP1A2 and CYP3A4, and among which CYP2E1 andCYP1A2 are the major enzymes for metabolism. The metabolite produced bythese enzymes, N-acetyl-p-benzoquinoneimine (NAPQI) is a very activeelectrophile. Under normal conditions, NAPQI will react immediately withglutathione in the cell and form non-toxic mercaptide. Overdose ofacetaminophen makes the consumption rate of glutathione greater than itssynthesis rate and when the glutathione level of the cell is lower thanthe normal range of 30%, NAPQI will bind to large molecules or nucleicacids containing cysteine and lead to liver damage. From histochemicalstains, NAPQI will bind to the thiol group of cysteine and form acovalent bond in centrilobular areas before occurrence of liver cellnecrosis.

Patients with liver disease, alcohol addiction or who are taking drugswhich may induce the activity of P450 such as carbamazepine, ethanol,Isoniazid, Phenobarbital (may be other barbiturates), Phenytoin,Sulfinpyrazone, Sulfonylureas, Rifampin and Primidone are thesusceptible groups of developing severe hepatotoxicity caused by APAPand may easily die if the patient also develops complications such asadult respiratory distress syndrome, cerebral edema, uncontrollablebleeding, infection or Multiple organ dysfunction syndrome (MODS). Takealcohol for example, alcohol is mainly eliminated by CYP2E1 of liver andits mechanism of APAP intoxication is divided into three stages: at thefirst stage alcohol competes the receptors for CYP2E1 with APAP in theliver and the concentration of NAPQI will reduce during the stage, atthe second stage alcohol prolongs the half-life of CYP2E1 from 7 hoursto 37 hours which increases the level of CYP2E1 in the liver and theconcentration of NAPQ1 will slowly increase during this stage, and atthe third stage, during alcohol withdrawal, more CYP2E1 is found in theliver to eliminate acetaminophen and consequently the toxic metabolitesof acetaminophen increases significantly and lead to liver damage.Recent studies have shown that diallyl sulfide can effectively preventhepatotoxicity caused by acetaminophen in mice and further demonstrateddiallyl sulfide can inhibit the activity of CYP2E1. It is speculatedthat the protection mechanism of diallyl sulfide against hepatotoxicityinduced by acetaminophen is by inhibition of the production of theintermediate NAPQI from acetaminophen. Previous studies have suggestedby inhibition the consumption of reduced glutathione in liver cells,oxidation activation, mitochondrial dysfunction and DNA damage caused byNAPQI can be reduced and subsequently minimize liver damage induced byacetaminophen. For example, Panax notoginseng, adenosine and itsderivatives adenosine monophosphate, adenosine diphosphate and adenosinetriphosphate can prevent liver damage induced by acetaminophen throughthis protection mechanism.

Fatty liver is considered another factor leading to liver damages. Undernormal circumstances, fat accounts for 3% by weight of the liver.Clinically, “fatty liver disease (FLD)” means fat in the liver exceeds5% by weight of the liver, or more than 10% of the liver cells showvesicular fatty changes in the liver tissue sections. According to thecauses of diseases, fatty liver can be divided into alcoholic fattyliver diseases (AFLD), non-alcoholic fatty liver diseases (NAFLD), orother fatty liver diseases derived from other factors, such as drugs.Fatty liver diseases are pathologically characterized by the appearanceof fatty metamorphosis or steatosis, steatohepatitis, or the like. Bythe percentage of liver cells suffering from steatosis, fatty liver iscategorized as mild (<33%), moderate (33-66%) and severe (>66%).Previously, fatty liver was considered a benign and reversiblecondition, and thus less taken seriously, but recent studies had foundthat it will lead to severe liver fibrosis and cirrhosis, and even livercancer. As the population of obese people increases, the prevalence ofFLD also increases.

The main cause of liver diseases in European and American countries isdue to chronic excessive drinking, therefore, the vast majority of liverdiseases are caused by alcohol lesions. But over the past 15-20 years,NAFLD has become the first cause of diseases to be considered for liverdysfunction in European and American countries. Thaler had everdescribed NAFLD in 1962. In 1980, Ludwig proposed “Non-alcoholicsteatohepatitis (NASH)” from accompanying NAFLD he found in a group ofobese female patients with diabetes and hyperlipidemia. Thereafter, in1986, Schaffner emphasized again that NASH played an important role inthe mechanism of fibrosis derivation in the course of NAFLD. Until 1998,Day found that 15-50% of patients with NASH were suffered from differentdegrees of fibrosis derivation, so clinicians started to pay attentionto NAFLD. Today, in addition to AFLD, NASH is not just a stage in thenatural progression of NAFLD in clinical practice. Due to the presenceof NASH, NAFLD is no longer considered a benign liver disease.

Regarding the mechanism of NAFLD, Day and James in the United Kingdomproposed Two-hit hypothesis based on a large number of clinicalresearches and animal experiments. Fatty liver occurs upon the firsthit, and steatohepatitis occurs upon the second hit. The first hit isprompted by excessive accumulation of fat in the liver, which is causedby obesity, hyperlipidemia, etc. The second hit is due to oxidativestress and the effect of reactive oxygen species (ROS) in mitochondria,resulting in lipid peroxidation on the liver cell membrane, release oforiginal inflammatory cytokines and free radicals, and fibrosis due toactivation of stellate cells, and leading to liver cell necrosis. Themechanism of NASH involves the peroxidation of triglyceride, oxidativestress, ROS response, increased peroxidation of lipids in liver cells,or increase of cytokines and liver enzymes, leading to a series ofautoimmune interactions.

The causes of fatty liver are mostly associated with long-term excessiveintake of animal fat, protein, carbohydrates, excess caloriestransforming into fat accumulated in the body, leading to obesity andfatty liver. Patients with fatty liver may have normal blood GOT/GPTvalues. Therefore, a correct diagnosis of fatty liver must use theabdominal ultrasound, which currently provides more than 97% accuracy.

Currently, there is no ideal drug providing specific therapeutic effectsfor FLD, the treatment guidelines of which aim at improving thepotential risk factors or controlling the progress of chronic diseasesby using drugs. It is recommended to apply symptomatic treatmentsaccording to the causes of fatty liver. For example, those who sufferingfrom fatty liver caused by overweight should lose weight moderately.Anyone with alcoholic fatty liver needs to quit drinking and eats abalanced diet for improving the conditions. Chemicals or drugs thatdamage liver and lead to fatty liver diseases through long-term contactshall immediately be stopped using. Fatty liver caused by diseases, suchas hepatitis C, high blood fat, etc., shall be treated by treating theoriginal diseases, such as treating hepatitis C or controlling bloodlipids. However, if excessive triglycerides are due to personallyphysical factors, it is hard to ameliorate fatty liver diseases bylosing weight.

However, the current drugs that are commonly used in clinical to lowerserum triglycerides and cholesterol are often accompanied with sideeffects, for example, hepatotoxicity, myopathy such as myalgia,myositis, rhabdomyolysis, and the like. Regarding the lipid-loweringdrugs, muscle toxicity is the most notable side effect. Especially,Statins shows the highest occurrence of muscle toxicity, and fibric acidfollows. In addition, the lipid-lowering drugs have a “fat driving”effect, which “drives” blood lipids to the liver, where fat accumulationalready exists and the influx of lipids is difficult to be processed,leading to excessive accumulation of fat in the liver and making fattyliver worse. It can be seen that the lipid-lowering drugs are notsuitable for the treatment of FLD.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides new compounds, thestructure of which is represented by Formula (I) as follows

-   -   wherein    -   L is a saturated or unsaturated aliphatic group;    -   R is selected from the group consisting of hydrogen, a polyol        group and a saccharide group of (G)_(p) wherein G is a        monosaccharide residue and p is an integer from 1 to 100 wherein        at least one of the hydroxyl groups in (G)_(p) is substituted by        a halogen atom; and    -   Q is an integer from 2 to 4, and each of R is the same or        different,        or a pharmaceutically acceptable salt thereof.

In some embodiments, the compounds of the present invention arerepresented by Formula (II) as follows:R₁—O—X—(CH₂)_(m)—X—O—R₂  Formula (II),

-   -   wherein    -   X is C═O;    -   R₁ and R₂ are the same or different, selected from the group        consisting of hydrogen, a polyol group and a saccharide group of        (G)_(p) wherein G is a monosaccharide residue and p is an        integer from 1 to 100 wherein at least one of the hydroxyl        groups in (O_(p) is substituted by a halogen atom, wherein when        R₁ is hydrogen, then R₂ is not hydrogen; and    -   m is an integer from 1 to 40.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising at least one of the compounds as described hereinor a pharmaceutically acceptable salt thereof together with apharmaceutically acceptable carrier.

In still another aspect, the present invention provides a treatmentmethod by administering to a subject in need an effective amount of atleast one of the compounds as described herein or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the method of the present invention is provided toprevent or treat a disease or condition characterized by increasedcytochrome P450 activities or increased free radical levels in a subjectin need thereof.

In some embodiments, the method of the present invention is provided toprevent or treat organ injuries in a subject in need.

In some embodiments, the method of the present invention is provided toprevent or treat hepatotoxicity in a subject in need.

In some embodiments, the method of the present invention is provided toprevent or treat fatty liver, protect liver function or ameliorate liverdiseases caused by fatty liver or other associated disorders.

In yet another aspect, the present invention provides use of thecompounds as described herein or a pharmaceutically acceptable saltthereof for manufacturing a medicament. In particular, the medicament isuseful in preventing or treating (i) a disease or conditioncharacterized by increased cytochrome P450 activities or increased freeradical level, (ii) organ injuries, and/or (iii) hepatotoxicity, and/or(iv) preventing or treating fatty liver, protecting liver function orameliorating liver diseases caused by fatty liver or other associateddisorders.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following detailed description ofseveral embodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. For the purpose of illustrating the invention,there are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention is notlimited to the precise arrangements and instrumentalities shown.

In the drawings:

FIG. 1 shows the percentage of pro-drug remain or its relatedmetabolites formation in blood (in vitro).

FIG. 2 shows the plasma concentration vs. time profile for pro-drug andsucralose after oral administration of pro-drug in SD-rats.

FIG. 3 shows the plasma concentration vs. time profile for mannitolafter oral administration of pro-drug in SD-rats.

FIG. 4 shows the H&E staining results of liver tissues in animals. (A)the normal control, (B) the control group of APAP-induced liverinjuries, (C) the positive control group of treatment with NAC, (D) theexperimental group of treatment with mannitol (1.67 mg/kg), (E) theexperimental group of treatment with sucralose (1.67 mg/kg), (F) theexperimental group of treatment with mannitol (2.51 mg/kg) plussucralose (2.51 mg/kg), (G) the experimental group of treatment withmannitol (3.34 mg/kg) plus sucralose (3.34 mg/kg), and (H) theexperimental group of treatment with NAC and a combination of mannitol(3.34 mg/kg) and sucralose (3.34 mg/kg).

FIG. 5 shows liver tissue sections taken from mice that were inducedfatty liver, and then treated with different test compounds by groupsfor four weeks.

FIG. 6 shows a general scheme of synthesis process of the compound ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as is commonly understood by one of skill in theart to which this invention belongs.

As used herein, the articles “a” and “an” refer to one or more than one(i.e., at least one) of the grammatical object of the article. By way ofexample, “an element” means one element or more than one element.

I. Compounds

In one aspect, the present invention provides new compounds, thestructure of which is represented by Formula (I) as follows

-   -   wherein    -   L is a saturated or unsaturated aliphatic group;    -   R is selected from the group consisting of hydrogen, a polyol        group and a saccharide group of (G)_(p) wherein G is a        monosaccharide residue and p is an integer from 1 to 100 wherein        at least one of the hydroxyl groups in (G)_(p) is substituted by        a halogen atom; and

-   Q is an integer from 2 to 4, and each of R is the same or different,    or a pharmaceutically acceptable salt thereof.

The term “aliphatic” or “aliphatic group”, as used herein, denotes ahydrocarbon moiety that may be straight-chain (i.e., unbranched),branched, or cyclic (including fused, bridging, and spiro-fusedpolycyclic) and may be completely saturated or may contain one or moreunits of unsaturation, but which is not aromatic. In general, aliphaticgroups contain 1-40 carbon atoms. In some embodiments, aliphatic groupscontain 1-20 carbon atoms, or 1-12 carbon atoms, 1-8 carbon atoms, or1-4 carbon atoms. In some embodiments, aliphatic groups contain 3-20carbon atoms, or 3-12 carbon atoms, 3-8 carbon atoms, or 3-4 carbonatoms. Suitable aliphatic groups include, but are not limited to, linearor branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereofsuch as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

In certain embodiments, the L group in Formula (I) is selected from (a)a straight alkyl group, (b) a branched alkyl group, (c) a straight alkylgroup substituted with a benzene ring, (d) a branched alkyl groupsubstituted with a benzene ring, (e) a benzenyl group where the benzenering contains a straight chain aliphatic group, and (f) a benzenyl groupwhere the benzene ring contains a branch chain of aliphatic group.

The term “polyol group”, as used herein, denotes an alcohol containingmultiple hydroxyl groups (two or more hydroxyl groups) per molecule. Inparticular, the polyol group can be linear or circular, substituted orunsubstituted, or mixtures thereof, so long as the resultant complex iswater-soluble and pharmaceutically acceptable.

In some embodiments, the polyol group is a C3-24 polyol, particularly, aC3-20 polyol, more particularly, a C3-12 polyol, or a C3-12 polyol,containing 2 or more hydroxyl groups.

In more particular embodiments, the polyol group is represented by—CH—(CHOH)_(n)CH₂OH wherein n is 1-22, 1-18, 1-10, or 1-6. In onecertain example, n is 4.

Preferred polyols are sugar alcohols. Examples of polyols include, butare not limited to, 3-carbon polyols (e.g. glycerol, erythritol andthreitol); 5-carbon polyols (e.g. arabitol, xylitol and ribitol);6-carbon polyols (e.g. mannitol, sorbitol, galactitol, fucitol, iditoland inositol); 12-carbon polyols (e.g. volemitol, isomalt, maltitol andlactitol); 18-carbon polyols (e.g. maltotriitol); and 24-carbon polyols(maltotetraitol).

In Formula (I), G represents a monosaccharide residue. Themonosaccharide as used herein is preferably a 6-carbon monosaccharidehaving the chemical formula C₆H₁₂O₆ (i.e. hexose). The hexose may be inthe D configuration, the L configuration, or a combination thereof.Hexoses are typically classified by functional groups. For example,aldohexoses have an aldehyde at position 1 such as allose, altrose,glucose, mannose, gulose, idose, galactose, and talose; and ketohexoseshave a ketone at position 2 such as psicose, fructore, sorbose, andtagatose. A hexose also contains 6 hydroxyl groups and the aldehyde orketone functional group in the hexose may react with neighbouringhydroxyl functional groups to form intramolecular hemiacetals orhemiketals, respectively. If the resulting cyclic sugar is a 5-memberedring, it is a furanose. If the resulting cyclic sugar is a 6-memberedring, it is a pyranose. The ring spontaneously opens and closes,allowing rotation to occur about the bond between the carbonyl group andthe neighbouring carbon atom, yielding two distinct configurations (αand β). The hexose may be in either the S configuration or the Rconfiguration.

According to the present invention, at least one of the hydroxyl groupsin the one or more monosaccharide residues in formula (I) is substitutedby a halogen atom. Examples of the halogen atom includes chlorine,bromine and iodine. Specifically, the halogen atom is chlorine.

As used herein, the term “S” or “R” is a way to name an optical isomerby its configuration, without involving a reference molecule, which iscalled the R/S system. It labels each chiral center R or S according toa system by which its ligands are each assigned a priority, according tothe Cahn Ingold Prelog priority rules, based on atomic number. Thissystem labels each chiral center in a molecule (and also has anextension to chiral molecules not involving chiral centers). If thecompound has two chiral centers, it can be labeled, for example, as an(S,S) isomer versus an (S,R) isomer.

As used herein, the term “pharmaceutically acceptable salt” includesacid addition salts. “Pharmaceutically acceptable acid addition salts”refer to those salts which retain the biological effectiveness andproperties of the free bases, which are formed with inorganic acids suchas hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, pyruvic acid, maleic acid, malonic acid, succinic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, trifluoroacetic acid and the like.

In some embodiments, in Formula (I), q is 2, 3 or 4, at least one of theR group is different from another one of R.

In certain embodiments, in Formula (I), q is 2.

In such embodiments, the compound of the present invention can berepresented by Formula (II) as follows:R₁—O—X—(CH₂)_(m)—X—O—R₂  Formula (II),

-   -   wherein    -   X is C═O;    -   R₁ and R₂ are the same or different, selected from the group        consisting of hydrogen, a polyol group and a saccharide group of        (G)_(p) wherein G is a monosaccharide residue and p is an        integer from 1 to 100 wherein at least one of the hydroxyl        groups in (G)_(p) is substituted by a halogen atom, wherein when        R₁ is hydrogen, then R₂ is not hydrogen; and    -   m is an integer from 1 to 40.        or a pharmaceutically acceptable salt thereof.

In certain embodiments, in Formula (II), R₁ is the polyol group and R₂is the saccharide group of (G)_(p). In such case, the compound ofFormula (II) is deemed as a conjugate of the polyol moiety linked to thesugar moiety by a linker via ester bonds. In particular, the linker isrepresented by —O—X—(CH₂)_(m)—X—O— (Formula (L)) wherein X is C═O and mis 1-40, 1-20, 1-12, 1-8 or 1-4, more particular, m is 3-20, 3-12, 3-8or 3-4. In one certain example, m is 4.

In some embodiments, p is 2. The saccharide group is represented by-G₁-O-G₂, wherein G₁ and G₂ are the same or different, selected from thegroup consisting of an aldohexose and a ketohexose, and at least one ofthe hydroxyl groups in G₁ or at least one of the hydroxyl groups in G₂is substituted by a halogen atom.

In some embodiments, G₁ is glucose wherein one of the hydroxyl groups issubstituted by chlorine; and G₂ is fructose wherein two of the hydroxylgroups are substituted by chlorine.

In certain embodiments, the saccharide group is represented by formula(Ia)

Certain examples of the compound of the present invention are asfollows:((2R,3R,4R,5R,6R)-6-(((2R,5R)-2,5-bis(chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-chloro-4,5-dihydroxytetrahydro-2H-pyran-2-yl)methyl((2R,3R,4R)-2,3,4,5,6-pentahydroxyhexyl) adipate

and

-   C6-mannitol of Formula 2

In another aspect, the present invention provides an intermediate ofFormula C as follows:

wherein Ph is phenyl and Bn is benzyl.

The compound of Formula (I) can be chemically synthesized for example bya process as shown in the general scheme of FIG. 6.

In particular, a linker agent that can provide one or more —COOH groupto perform esterification with an alcohol is provided. In step 1, thelinker agent providing a first —COOH group (others if available areprotected) reacts with R having a first free hydroxyl group (others ifavailable are protected) to proceed with the first esterification,producing the compound of Formula (I) where q is 1. In step 2, thelinker agent providing a second —COOH group (others if available areprotected) reacts with R having a second free hydroxyl group (others ifavailable are protected) to proceed with the second esterification,producing the compound of Formula (I) where q is 2. In step 3, thelinker agent providing a third —COOH group (others if available areprotected) reacts with R having a third free hydroxyl group (others ifavailable are protected) to proceed with the third esterification,producing the compound of Formula (I) where q is 3. In step 4, thelinker agent providing a fourth —COOH group (others if available areprotected) reacts with R having a fourth free hydroxyl group (others ifavailable are protected) to proceed with the third esterification,producing the compound of Formula (I) where q is 4.

In some embodiments, the linker agent to perform the esterification isrepresented by Formula (La)P₁—O—X—(CH₂)_(m)—X—O—P₂  Formula (La)wherein X and m are as defined above, and P₁ and P₂ are the same ordifferent and are a protecting group or H.

In some embodiments, the linker agent to perform the esterification isrepresented by Formula (La)

As used herein, a “protecting group” is a chemical group that isattached to a functional moiety (for example to the oxygen in a hydroxylgroup or the nitrogen in an amino group, replacing the hydrogen) toprotect the functional group from reacting in an undesired way. Aprotecting group includes, for example, t-butyl group, a cycloalkylgroup (e.g., cyclohexyl group), an aryl group (e.g., 2,4-dinitrophenylgroup), an aralkyl group (e.g., benzyl group, 2,6-dichlorobenzyl group,3-bromobenzyl group, 2-nitrobenzyl group, 4-dimethylcarbamoylbenzylgroup, and triphenylmethyl group), a tetrahydropyranyl group, an acylgroup, an alkoxycarbonyl group (e.g., t-butoxycarbonyl group), anaralkyloxycarbonyl group (e.g., benzyloxycabonyl group,2-bromobenzyloxycarbonyl group), a dialkylphosphinothioyl group (e.g.,dimethylphosphinothioyl group) and a diarylphosphinothioyl group (e.g.,diphenylphosphinothioyl group). A preferred protecting group includes anacyl group and the like.

In one certain example, Scheme 1 is provided in Example 1 showing theparticular synthesis process of the compound of the present invention.

II. Uses of the Compounds of the Present Invention

The compounds of the invention can be used as a medicament for treatmentmethods. In general, the compound of Formula (I) acts as a prodrug thatafter administration can turn into metabolites providing therapeuticeffects as needed as described herein. In one example, the compound ofFormula (I) is compound F, which after administration can turn intomannitol, sucralose and C6-mannitol, all of which can act as P450inhibitors and provide anti-hepatotoxicity effects, for example. Seeexamples below.

The present invention provides a treatment method by administering to asubject in need an effective amount of at least one of the compounds asdescribed herein or a pharmaceutically acceptable salt thereof.

It is found that compounds of the invention are effective as P450inhibitors, for example.

In some embodiments, the method of the present invention is provided toprevent or treat a disease or condition characterized by increasedcytochrome P450 activities in a subject in need thereof.

Examples of such diseases or conditions are listed in Table A.

TABLE A Diseases alcoholic hepatitis hepatoblastoma drug-inducedhepatitis Liver, renal chronic disease alcoholic liver cirrhosis obesityliver disease poisoning liver cirrhosis insulin resistance alcohol abusechronic liver disease isoniazid toxicity hepatitis chronic nonalcoholicsteatohepatitis renal disease tuberculosis inflammation Hepatitisalcohol withdrawal Fatty liver disease alcoholic cirrhosisHepatocellular carcinoma liver damage liver diseases alcoholicalcoholism hepatitis halothane hepatitis toxic fatty liver alcoholicfatty liver hepatic necrosis alcohol-related disorders cirrhosiscerebrovascular disease acute alcoholic hepatitis coronary arterydisease Liver, renal histopathology Liver, renal cell damageEthanol-induced and obesity-induced oxidant stress and liver injuryLiver, renal necrosis heavy metal poisoning hepatitis c chronic liverfibrosis cardiovascular disease atherosclerosis

In some embodiments, the method of the present invention is provided toprevent or treat a disease or condition characterized by increased freeradical levels in a subject in need thereof.

In some embodiments, the method of the present invention is provided toprevent or treat organ injuries in a subject in need.

In particular examples, the organ injuries are in liver or kidney.

In particular examples, organ injuries or hepatotoxicity are caused by atherapeutic drug, CCl₄ or lipid accumulation.

In particular examples, the therapeutic drug is acetaminophen.

In some embodiments, the method of the present invention is provided toprevent or treat hepatotoxicity in a subject in need.

In some embodiments, the method of the present invention is provided toprevent or treat fatty liver, protecting liver function or amelioratingliver diseases caused by fatty liver or other associated disorders.

As used herein, the term “liver fat content” refers to the content offat that is accumulated in the liver of a subject and includes broadlydefined lipids, such as triglyceride (TG) and cholesterol. As usedherein, the term “reducing liver fat content” generally refers to thereduction of the content of abnormal liver fat in a subject, i.e. todecrease the content of abnormal liver fat and, more particularly, tolower the content of abnormal liver fat to normal level. For example,under normal circumstance, fat accounts for 3% by weight of the liver.If fat in the liver exceeds 5% by weight of the liver, it is determinedas abnormal fat accumulation (the liver fat content described above is arelative percentage for exemplification, and may vary due to ethnicityand other factors). In a specific aspect, the term “reducing liver fatcontent” used herein could means that the content of abnormal liver fatin a subject is reduced, for example, from 5% by weight of the liver ormore to 3% by weight of the liver. Liver fat content can be assessed bystandard analytical methods, including but not limited to ultrasoundanalysis, magnetic resonance imaging (MRI), magnetic resonancespectroscopy (MRS), computed tomography (CT), and liver biopsy.

As used herein, the term “liver function” refers to one or morephysiological functions performed by the liver. Liver function can beanalyzed by a lot of conventional assays, such as alanineaminotransferase (ALT) analysis or aspartate transaminase (AST)analysis. According to the present invention, the compound describedherein can be used to maintain the liver function, including improvementof the liver function and preventing the liver from damage.

As used herein, the term “liver diseases” refers to liver cell injury ordamage caused by certain factors, which then potentially lead to liverdysfunction. According to the present invention, the compound proposedherein can be used to ameliorate liver diseases caused by fatty liver insome embodiments. More particularly, “liver damage” used herein refersto liver with histological or biochemical dysfunction, as compared withnormal liver. In a specific embodiment, “liver damage” refers to liverlesions caused by alcoholic or non-alcoholic factors, such as high fatdiet or obesity, or therapeutic drugs or organic solvents. In a specificembodiment, “liver damage” can be liver tissue damage with one or morecharacteristics selected from steatosis, lobular inflammation,hepatocyte ballooning, and vesicular fat droplets produced by livercells. In a specific embodiment, “liver damage” can be biochemicaldysfunction of liver, which can be determined from the activity ofalanine aminotransferase (ALT) or aspartate transaminase (AST). Higheractivity of ALT or AST indicates severer dysfunction of liver'sbiochemical function.

As used herein, the term “liver antioxidant activity” refers to theactivity or ability against oxidative stress. Improvement of liverantioxidant activity of a subject by the compound according to thepresent invention refers to, includes, but is not limited to reducingoxidative stress or enhancing enzyme activity or content of the membersof antioxidant systems. The members of antioxidant systems may beglutathione peroxidase (GPx), glutathione (GSH), glutathione reductase(GRd), and/or superoxide dismutase (SOD).

According to the present invention, the compound described hereinincludes common excipients and bioflavonoids, which may be used toreduce liver fat content and ameliorate associated disorders. The term“associated disorders” described herein includes the disorders caused byabnormal accumulation of liver fat and including, but not limited tofatty liver diseases, acute and chronic alcoholic fatty liver diseases,acute and chronic non-alcoholic fatty liver diseases, acute and chronicalcoholic hepatitis, acute and chronic non-alcoholic steatohepatitis,non-alcoholic cirrhosis and alcoholic cirrhosis (ICD-9-CM DiagnosisCodes: 571.8, 571.0, 571.1, 571.2, 571.3, 571.4, 571.5, 571.9).

As used herein, the term “preventing” refers to the preventive measuresfor a disease or the symptoms or conditions of a disease. The preventivemeasures include, but are not limited to applying or administering oneor more active agents to a subject who has not yet been diagnosed as apatient suffering from the disease or the symptoms or conditions of thedisease but may be susceptible or prone to the disease. The purpose ofthe preventive measures is to avoid, prevent, or postpone the occurrenceof the disease or the symptoms or conditions of the disease.

As used herein, the term “treating” refers to the therapeutic measuresto a disease or the symptoms or conditions of a disease. The therapeuticmeasures include, but are not limited to applying or administering oneor more active agents to a subject suffering from the disease or thesymptoms or conditions of the disease or exacerbation of the disease.The purpose of the therapeutic measures is to treat, cure, mitigate,relieve, alter, remedy, ameliorate, improve, or affect the disease, thesymptoms or conditions of the disease, disability caused by the disease,or exacerbation of the disease.

As used herein, a “CYP2E1 inhibitor” is any compound, substance ormaterial that can inhibit CYP2E1 activity. A number of assays areavailable for analysis of the CYP2E1 activity such as a human or ratliver microsome assay.

As used herein, a subject in need of the treatment according to theinvention includes human and non-human mammals. Non-human mammalsinclude, but are not limited to, companion animals such as cats, dogsand the like and farm animals such as cattle, horses, sheep, goats,swine and the like.

The term “effective amount” or the like refers to that amount of anactive agent sufficient to achieve a desired therapeutic, prophylactic,and/or biological effect in a subject, such as reducing drug-inducedside effects, or prohibiting, improving, alleviating, reducing orpreventing one or more symptoms or conditions or progression of adisease. The actual effective amount may change depending on variousreasons, such as administration route and frequency, body weight andspecies of the individual receiving said pharmaceutical, and purpose ofadministration. Persons skilled in the art may determine the dosage ineach case based on the disclosure herein, established methods, and theirown experience.

The term “a standard dose” as used herein refers to an effective dose ofa therapeutic agent that is recommended by authoritative sources in thepharmaceutical community including the Food and Drug Administration andoften used in routine practice. The term “a reduced dose” as used hereinrefers to a dose that is lower than a standard dose but still retainssubstantially the same therapeutic effects of the same therapeuticagent. Specifically, according to the invention, a reduced dose of atherapeutic drug is about 90% or less, 80% or less, 70% or less, 60% orless, 50% or less, of standard therapeutic dose of the therapeutic drug.

In some embodiments, an effective amount of active ingredients as usedherein may be formulated with a pharmaceutically acceptable carrier intoa pharmaceutical composition of an appropriate form for the purpose ofdelivery and absorption.

As used herein, “pharmaceutically acceptable” means that the carrier iscompatible with the active ingredient in the composition, and preferablycan stabilize said active ingredient and is safe to the individualreceiving the treatment. Said carrier may be a diluent, vehicle,excipient, or matrix to the active ingredient. The composition mayadditionally comprise lubricants; wetting agents; emulsifying andsuspending agents; preservatives; sweeteners; and flavoring agents. Thecomposition of the present invention can provide the effect of rapid,continued, or delayed release of the active ingredient afteradministration to the patient.

According to the present invention, the form of said composition may betablets, pills, powder, lozenges, packets, troches, elixers,suspensions, lotions, solutions, syrups, soft and hard gelatin capsules,suppositories, sterilized injection fluid, and packaged powder.

The composition of the present invention may be delivered via anyphysiologically acceptable route, such as oral, parenteral (such asintramuscular, intravenous, subcutaneous, and intraperitoneal),transdermal, suppository, and intranasal methods. Regarding parenteraladministration, it is preferably used in the form of a sterile watersolution, which may comprise other substances, such as salts or glucosesufficient to make the solution isotonic to blood. Preparation of anappropriate parenteral composition under sterile conditions may beaccomplished with standard pharmacological techniques well known topersons skilled in the art, and no extra creative labor is required.

In certain embodiments, the compound of Formula (I) of the presentinvention or a pharmaceutically acceptable salt thereof can be used inpreventing or treating injuries in organs e.g. in liver or kidney, whichmay be caused by overdose of therapeutic drugs (e.g. acetaminophen) orexposure of alcohol, a chemical agent, a biomolecule or any substancethat may cause toxic effects in these organs.

Specifically, injuries in liver may include injuries, damages or loss ofhepatic cells or tissues, leading to abnormal liver functions orcontents of liver proteins. In some embodiments, the liver injuries asdescribed herein are acute liver injuries which mean liver injuries ofrelatively rapid onset e.g. less than 12 week, particularly less than 6weeks duration from time of onset of symptoms. In some embodiments,patients with acute liver injuries are with no background of chronichepatic diseases.

Specifically, injuries in kidney may include injuries, damages or lossof renal cells or tissues, leading to abnormal renal functions. Suchrenal injuries may be identified, for example, by a decrease inglomerular filtration rate, a reduction in urine output, an increase inserum creatinine, an increase in serum cystatin C, etc. In someembodiments, the renal injuries as described herein are acute renalinjuries, which may mean an abrupt or rapid decline in renal filtrationfunction, for example, within 14 days, preferably within 7 days, morepreferably within 72 hours, and still more preferably within 48 hours.

In one particular embodiment, the compound of Formula (I) of the presentinvention or a pharmaceutically acceptable salt thereof is capable ofpreventing or treating an undesired condition caused by NAPQI(N-acetyl-p-benzoquinone imine).

Therefore, the present invention provides use of the compound of Formula(I) of the present invention or a pharmaceutically acceptable saltthereof for manufacturing a medicament for preventing or treating anundesired condition caused by NAPQI (N-acetyl-p-benzoquinone imine) in asubject. The present invention also provides a method for preventing ortreating an undesired condition caused by NAPQI (N-acetyl-p-benzoquinoneimine) in a subject in need, comprising administering to the subject thecompound of Formula (I) of the present invention or a pharmaceuticallyacceptable salt thereof in an amount effective to prevent or treat theundesired condition.

III. Combined Use of Compound of the Present Invention with Other ActiveAgent

The compound of the present invention and/or its metabolites can beadministered in combination with one or more additional active agents,particularly those acting as P450 inhibitors and/or providinganti-hepatotoxicity activities and/or those with anti-fatty liveractivities, so as to provide a synergistic effect, for example.

Some active agents acting as P450 inhibitors (named “a first activeagent(s)”) are described in PCT/CN2013/087049 (U.S. Ser. No. 14/441,317,the content of which is hereby incorporated by reference in itsentirety). Particular examples of such P450 inhibitors include but arenot limited to polyethylene glycol sorbitan monolaurate (Tween 20),microcrystalline cellulose, dicalcium phosphate dihydrate, Brij 35,saccharin, mannitol, Cremophor RH40, sucralose, crospovidone, sodiumstarch glycolate, Eudragit S100, croscarmellose sodium, Pluronic F68,menthol, low-substituted hydroxypropyl cellulose, pregelatinized starch,Dextrates NF hydrated, citric acid, Cremophor EL, Aerosil 200, Myrj 52,sorbic acid, lemon oil, hydroxypropyl cellulose, Sorbitol, acesulfamepotassium, hydroxypropyl methylcellulose, lactose monohydrate,maltodextrin, Brij 58, Brij 76, Tween 80, Tween 40, PEG 400, PEG 4000,PEG 8000, Span 60, sodium benzoate, hydroxy ethylmethylcellulose,methylcellulose, Span 80, sodium cyclamate, glyceryl behenate, oxidered, glycerin monostearate, Copovidone K28, starch acetate, magnesiumstearate, sodium lauryl sulfate, Providone K30, PEG 2000, andN-acetylcysteine (NAC) and any combination thereof.

In certain embodiments, the one or more first active agents to be usedin combination with the compound of Formula (I) of the present inventionare selected from the group consisting of dicalcium phosphate dehydrate,menthol, mannitol, sucralose, N-acetylcysteine (NAC) and any combinationthereof.

Some active agents with anti-fatty liver activities (named “a secondactive agent”) are described in PCT/CN2016/078039, the content of whichis hereby incorporated by reference in its entirety. Particular examplesof active agents with anti-fatty liver activities include but are notlimited (ii) a second active agent selected from the group consistingof: sodium lauryl sulfate, menthol, sucralose, mannitol, sorbitol,saccharin, glycerin, sodium benzoate, oxide red, pregelatinized starch,sodium cyclamate, sorbic acid, lemon oil, citric acid, butylatedhydroxyanisole, poncirin, isovitexin, eriodictyol, ergosterol,β-myrcene, hyperoside, (+)-catechin, galangin, morin, sciadopitysin,didymin, gossypin, luteolin-7-glucoside, (+)-taxifolin, trans-cinnamicacid, diosmin, linarin, xylitol, luteolin, swertiamarin, puerarin,phloridzin, sinensetin, (−)-epigallocatechin, kaempferol, ursolic acid,silymarin, (+)-limonene, hesperidin, (−)-epicatechin-3-gallate, silybin,formononetin, myristic acid ethyl ester, eicosapentaenoic acid (EPA),wongonin, povidone K-30, protocatechuic acid, umbelliferone, hesperitin,nordihydroguaiaretic acid, neohesperidin, naringin, (−)-epicatechin,glycyrrhizin, baicalin, quercitrin, baicalein and any combinationsthereof.

In certain embodiments, the one or more second active agents to be usedin combination with the compound of Formula (I) of the present inventionare selected from the group consisting of sodium lauryl sulfate,menthol, sucralose, mannitol, sorbitol, saccharin, glycerin, sodiumbenzoate, oxide red, pregelatinized starch, sodium cyclamate, sorbicacid, lemon oil, citric acid, butylated hydroxyanisole, poncirin,isovitexin, eriodictyol, ergosterol, β-myrcene, hyperoside,(+)-catechin, galangin, morin, sciadopitysin, didymin, gossypin,luteolin-7-glucoside, (+)-taxifolin, trans-cinnamic acid, diosmin,linarin, xylitol, luteolin, swertiamarin, and any combinations thereof.

In certain embodiments, the one or more second active agents to be usedin combination with the compound of Formula (I) of the present inventionare selected from the group consisting of puerarin, phloridzin,sinensetin, (−)-epigallocatechin, kaempferol, ursolic acid, silymarin,(+)-limonene, hesperidin, (−)-epicatechin-3-gallate, silybin,formononetin, myristic acid ethyl ester, eicosapentaenoic acid (EPA),wongonin, povidone K-30, protocatechuic acid, umbelliferone, hesperitin,nordihydroguaiaretic acid, neohesperidin, naringin, (−)-epicatechin,glycyrrhizin, baicalin, quercitrin, baicalein and any combinationsthereof.

In certain embodiments, the one or more second active agents to be usedin combination with the compound of Formula (I) of the present inventionare selected from the group consisting of eriodictyol, mannitol,menthol, sucralose, saccharin, and any combinations thereof.

In certain embodiments, the one or more second active agents to be usedin combination with the compound of Formula (I) of the present inventionare selected from the group consisting of (1) a combination of saccharinand mannitol, (2) a combination of menthol and mannitol, (3) acombination of sucralose and mannitol, (4) a combination of eriodictyoland mannitol, (5) a combination of eriodictyol and sucralose, (6) acombination of menthol, mannitol, and eriodictyol, and (7) a combinationof sucralose, mannitol, and eriodictyol.

Specifically, the compound of Formula (I) or a pharmaceuticallyacceptable salt thereof and the one or more additional agents can beadministered simultaneously or sequentially.

In the present invention, it is further provided that the compound ofFormula (I) of the present invention or a pharmaceutically acceptablesalt thereof is capable of preventing or treating an undesired conditioncaused by NAPQI (N-acetyl-p-benzoquinone imine).

As a particular embodiments, the present invention provides acombination of the compound of Formula (I) and/or its metabolites withN-acetylcysteine (NAC). The prevent invention also provides a method foradministering N-acetylcysteine (NAC) in a subject in need, comprisingadministering to the subject NAC in combination with the compound ofFormula (I) and/or its metabolites. In one embodiment, the combinationor the method of the present invention is effective in preventing ortreating a disease or disorder for which NAC is effective. In someembodiments, the disease or disorder to be treated or prevented by NACis selected from the group consisting of Myoclonus Epilepsy, acuterespiratory distress syndrome, heavy metal poisoning, influenzainfection, heart disease, Sjogren's syndrome, chronic bronchitis,epilepsy (Unverricht-Lundborg type) and HIV infection.

The present invention is further illustrated by the following examples,which are provided for the purpose of demonstration rather thanlimitation.

EXAMPLES Example 1: Synthesis of Compound of Formula 1 (Compound F) ofthe Present Invention Synthetization of((2R,3R,4R,5R,6R)-6-(((2R,5R)-2,5-bis(chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-chloro-4,5-dihydroxytetrahydro-2H-pyran-2-yl)methyl((2R,3R,4R)-2,3,4,5,6-pentahydroxyhexyl) Adipate (Formula 1) (CompoundF)

The synthetic strategy for the synthesis of Formula 1 (Compound F) isshown in Scheme 1.

General Methods

All chemicals were obtained from commercial sources and used as receivedunless otherwise stated.

The chromatographic purity of products was assessed in a condition asfollows:

Mobile phase composition A: Methanol:H₂O = 5/95(v/v), Contain 0.05%NH₄OH B: Methanol:H₂O = 95/5(v/v), Contain 0.05% NH₄OH Chromatographysystem: Error! Not a valid link. Column type Waters ® Acquity UPLCHSST₃, 1.8 μm, 100 × 2.1 mm Autosampler temperature 4° C. Column oventemperature 45° C. Flow rate 0.35 mL/min Analysis time 10 min Injectionvolume 5 μL Retention time 4.8 min

The MS analysis was conducted in a condition as follows:

Mass spectrometer settings: Mass spectrometer Triple Quadrupole MS (APIQtrap5500) Applied Biosystem, Inc. Detection MRM negative mode Pro-drug:m/z 688.9 → m/z 180.9

Bruker AMX-500 NMR spectrometer in MeOH-d₄ (δ_(H) 3.30, δ_(C) 49.0) orCDCl₃ (δ_(H) 7.24, δ_(C) 77.0) using Bruker's standard pulse program; inthe HMQC and HMBC experiments, Δ=1 s and J=140, 8 Hz, respectively, thecorrelation maps consisted of 512×1 K data points per spectrum, eachcomposed of 16 to 64 transients.

1.1 Mannitol (Compound (i)) to Compound (B)

1.1.1 Mannitol (Compound (i)) to Compound (i)-1

To a solution of D-mannitol (25 g, 0.137 mol) in DMF (250 mL) was addedbenzaldehyde (30 mL, 0.345 mmol) at r.t. under Ar. To the mixture wasadded concentrated sulfuric acid (10 mL) dropwise at 0° C. After beingallowed to warm up gradually to the r.t., the mixture was stirred for 3day. Then the mixture was poured into ice water (250 mL) and n-hexane(200 mL) under vigorous stirring. After the mixture was warm up to r.t.,the precipitate was filtered and washed with n-hexane. The precipitatewas suspended in chloroform and heated under reflux for 15 min undervigorous stirring. When the mixture reached r.t., the undissolvedprecipitate was collected and Recrystallization from EtOH gave desiredproduct as white solid (9.86 g, 20%). R_(f)=0.45 (EA/Hex=1/1).

1.1.2 Compound (i)-1 to Compound (i)-2

To a solution of 1,3,4,6-dibenzylidene (10 g, 27.9 mmol) in DMF (100 mL)was added benzyl bromide (7.96 mL, 66.96 mmmol) at r.t. under Ar. Themixture was cooled to 0° C. then 60% NaH (2.68 g, 66.96 mmol) was addedin few time. After being allowed to warm up gradually to the r.t., themixture was stirred for overnight. Then the reaction was quenched bywater (dropwise) and extracted with NaHCO_(3(aq)) and dichloromethane.The organic layer was dried with MgSO₄, concentrated in vacuum. Theresidue was purified by column chromatography on silica gel to afforddesired product. (10.39 g, 69%). R_(f)=0.2 (EA/Hex=1/6).

1.1.3 Compound (i)-2 to Compound (B)

To a solution of 2,5-dibenzyl-1,3,4,6-dibenzylidene (1.5 g, 2.78 mmol)in toluene (12.5 mL) was cooled to −18° C. (ice-salt bath). 1.2 M DIBALwas added (18.5 mL, 22.3 mmol) dropwise and warmed to r.t. After 1.5 h,the reaction was cooled to 0° C. then quenched by MeOH and 15%KOH_((aq)). The mixture was extracted with DCM, organic layer was drywith MgSO₄ and concentrated in vacuum. The residue was purify by columnchromatography on silica gel to afford desired product. (709 mg, 47%).R_(f)=0.1 (EA/HEX=1/5).

1.2 Sucralose (Compound (ii)) to Compound (D)

1.2.1 Compound (ii) to Compound (ii)-1

To a solution of sucralose (1 g, 2.5 mmol) in DCM (10 mL) was added HMDS(2.6 mL, 12.57 mmol) and TMSOTf (45 μL, 0.25 mmol). The reaction wasstirred for overnight in r.t. The reaction was concentrated in vacuumand pass through the cotton, wash by hexane. The filtrate wasconcentrated again in vaccum to get the product in quant. (1.9 g,quant.). R_(f)=0.9 (EA/HEX=1/8).

1.2.2 Compound (ii)-1 to Compound (D)

To a solution of penta-TMS sucralose (5 g, 6.6 mmol) in pyridine (150mL) was added 0.1 M pyridine-TsCl solution (6.6 mL) and stirred for 3days with open flask. The reaction was concentrate in vacuum andpurified by column chromatography on silica gel to afford desiredproduct. (1.4 g 30%). R_(f)=0.5 (EA/HEX=1/8).

1.3 Synthesis of6-Oxo-6-((2R,3R,4R)-2,3,4-Tris(Benzyloxy)-4-(2-Phenyl-1,3-dioxolan-4-yl)butoxy)hexanoicacid (compound (C))

In a flame dry R.B. flask compound A (165 mg, 1 eq.) was dissolved inDCM (5 mL) at 0° C., then to this was added pyridine (0.2 mL) and DMAP(50 mg). Reaction mixture was then stirred for 10 min, followed by Comp.B (59 mg, 1.5 eq.) was added. Reaction mixture was then stirred at roomtemperature for 5 h. TLC confirmed the completion of reaction. Reactionmixture was evaporated to dryness of rotavapour under reduced pressure.The crude compound was further purified by column chromatography toafford the desired compound as a colorless oil (136 mg, 67%).

1.4 Synthesis of((2R,3S,4R,5R,6R)-6-((2R,5R)-2,5-bis(chloromethyl)-3,4-bis((trimethylsilyl)oxy)tetrahydrofuran-2-yl)oxy)-3-chloro-4,5-bis((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)methyl((2R,3R,4R)-2,3,4-tris(benzyloxy)-4-(2-phenyl-1,3-dioxolan-4-yl) butyl)Adipate

To ice cold solution of compound C (100 mg, 1.0 eq.) in DCM was addedDCC (35 mg, 1.15 eq.) and stirred for 10 min. Then to this Compound D(112 mg, 1.2 eq.) and DMAP (5 mg, 0.25 eq. catalytic) was added.Reaction mixture was allowed to warm to rt and stirred for 4 hours. TLCconfirmed the completion of reaction. Reaction mixture was evaporated todryness on rotavapour under reduced pressure. The crude compound wasthen purified by column chromatography using neutral silica gel and 5 to15% ethyl acetate in Hexane with 1% Triethyl amine as an eluent toafford desired compound E as a colourless oil (84 mg, 42%).

1.5 Synthesis of((2R,3R,4R,5R,6R)-6-(((2R,5R)-2,5-bis(chloromethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-3-chloro-4,5-dihydroxytetrahydro-2H-pyran-2-yl)methyl((2R,3R,4R)-2,3,4,5,6-pentahydroxyhexyl) Adipate (Compound F)

In a flame dry Single neck R.B. flask compound E (500 mg, 1 eq.) wasdissolved in dry MeOH (20 mL), solution was then degassed by nitrogengas (Nitrogen gas syringe was deep inside the solution and Nitrogen waspurge for 15 min.). Then 10% Pd-C (200 mg, 33% w/w) was added cautiouslyto reaction mixture. Finally, reaction mixture was stirred underhydrogen balloon pressure for 6 hours. TLC confirmed the completion ofreaction. Reaction mixture was then filtered through celite bed and thebed was washed with dry methanol. The filtrate was evaporated to drynessof rotavapour under reduced pressure. Final compound was then kept underhigh vacuum to afford desired final compound F as colorless semisolid orwhite solid (190 mg, 73%). The structure of compound F were identifiedby high-resolution mass spectrophotometry and ¹³C NMR.

Example 2: Compound F as a Prodrug, Generating Metabolites whenIncubated with Blood (In Vitro)

2.1 Materials and Methods

Fresh human whole-blood were used for drug hydrolysis studies. Drug (10mg, compound F) was dissolved in 1 mL solution (20% methanol). Drughydrolysis (n=3) was performed in 20 mL of fresh whole-blood aliquotscontaining 1.0 mg of drug in a 50-mL flask thermostat at 37° C. in ashaking water bath. At time 0, the drug was added, and after varioustimes of incubation, the blood samples were collected at 0.25, 0.5,0.33, 0.75, 1, 2, 4, 6, 12 and 24 hrs. Blood sample were used 1 mLacetonitrile to quench the enzymatic hydrolysis of the drug as sampleswere obtained. Pro-drug and its related metabolites, such asC6-mannitol, mannitol and sucralose in blood were determined by An APIQTrap5500 triple-quadrupole mass spectrometer equipped with an ion-spray(ESI) source. The ESI interface was used in the negative-ion mode.

2.2 Results

The pro-drug was monitored at a transition of m/z 688.9→180.9, Sucralosewas monitored at a transition of m/z 395→4359; mannitol was monitored ata transition of m/z 452.3→4273.3; C6-mannitol was monitored at atransition of m/z 309→4101.1. All the compounds were identified byhigh-resolution mass spectrophotometry and ¹³C NMR. The structure ofC6-mannitol (formula (2)) is as follows:

The hydrolysis of pro-drug in blood was expressed by plotting thepercentage of Pro-drug remaining and the percentage of sucralose,mannitol and C6-mannitol increasing versus time after incubation of thepro-drug in blood (FIG. 1). The results shows that compound F acts as apro-drug which turns into its metabolites including sucralose, mannitoland C6-mannitol after incubated with blood in vitro.

Example 3: Pharmacokinetics Study in SD (Sprague Dawley)-Rats (In Vivo)

3.1 Materials and Methods

SD-rats were orally administered pro-drug at a dose of 3.67 mg/kg BW.Blood samples were collected into heparinized micro centrifuge tubes atintervals of 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Plasma samples wereimmediately obtained by centrifuging the blood samples at 8,000 rpm for10 min. The plasma samples were then stored at −80° C. until use. Theplasma samples were analyzed for pro-drug and its related metabolites,such as mannitol and sucralose by API QTrap5500 triple-quadrupole massspectrometer equipped with an ion-spray (ESI) source. The ESI interfacewas used in the negative-ion mode.

3.2 Results

The pro-drug was monitored at a transition of m/z 688.9→180.9, Sucralosewas monitored at a transition of m/z 395→359; mannitol was monitored ata transition of m/z 452.3→273.3; C6-mannitol was monitored at atransition of m/z 309→101.1.

FIG. 2 and FIG. 3 shows the plasma concentration time curves of pro-drugand its related metabolites, such as sucralose and mannitol in SD-ratswith single oral dosing of 3.67 mg/kg pro-drug, respectively. Theresults shows that compound F acts as a pro-drug which converts into itsmetabolites including sucralose, mannitol and C6-mannitol afteradministration in animals in vivo

Example 4: CYP2E1 Inhibitory Activity Assays

4.1 Materials and Methods

This example is preparation of microsomes from human liver for in vitroscreening of CYP450 isozyme inhibitors. Effective human hepatic CYP450isozyme inhibitors were tested and the principle for testing the CYP450isozyme inhibitors is based on the reaction of microsomal CYP450 isozymeprepared from the liver of different origin and its specific substrateChlorzoxazone (CZX). After addition of the test sample, the amount ofCYP450 isozyme metabolite standard 6-OH—CZX (6-Hydroxy-Chlorzoxazone) isspecific used for calculation of the CYP450 isozyme (CYP2E1) inhibitionratio of the test sample by using the amount of 6-OH—CZX of the controlgroup as the baseline.

All samples were tested in triplicate. To determine the percentageinhibition, each test compound was dissolved in 1, 2, 4 μg/mL to threedifferent concentrations. The CYP2E1 activity levels in the presence ofthe test compounds were compared with the control incubations. The500-μL reaction mixture, containing 0.5 mg of microsomal protein, wasincubated with 320 μM CZX in the presence of 5 mM MgCl₂ and 1 mM NADPHin 50 mM phosphate buffer with pH 7.4 at 37° C. for 30 min. The reactionwas terminated by ice-cold acetonitrile, and then 4-hydroxyl tolbutamidewas added as an internal standard. The organic phase was evaporated todryness and reconstituted into the mobile phase (methanol:water=1:1)prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS)analysis. An API 3000 triple-quadrupole mass spectrometer equipped withan ion-spray (ESI) source was used to determine 6-OH—CZX in the humanliver microsomes. The ESI interface was used in the positive-ion mode.The 6-OH—CZX was monitored at a transition of m/z 284.5→185.9.

Analysis of the results: convert the detected signal values obtainedfrom LC/MS/MS into the amount (pmol) of CYP450 isozyme metabolitestandard 6-Hydroxy-Chlorzoxazone using the control group as thebaseline, i.e. the CYP450 isozyme inhibition ratio of the control groupis 0%. The CYP450 isozyme activity levels in the presence of the testcompounds were compared with the control incubations.

4.2 Results

Diethyldithiocarbamic acid (DDTC) is a well-known inhibitor of CYP2E1.At a concentration of 100 μM, DDTC treatment resulted in 90.9%inhibition of CYP2E1 in human liver microsomes (measured using CZX as aCYP2E1 substrate). On the basis of the observed inhibitory activity ofDDTC, we tested the new compound (pro-drug) and its related metabolitesfor CYP2E1 inhibition at concentrations of 4, 2 and 1 μg/mL. The resultsas summarized in Table 1.

TABLE 1 The inhibition ratios of CYP2E1 inhibitors from in-vitroscreening of human liver microsomes Test compound CYP 2E1 inhibitionratio (%) Test concentration 4 μg/mL 2 μg/mL 1 μg/mL Control group 0 0 0Positive control (DDTC) (100 μM) (50 μM) (10 μM) 90.9 ± 0.8 51.2 ± 3.211.2 ± 2.4 Pro-drug 45.7 ± 2.5 33.3 ± 4.1 17.7 ± 0.7 Metabloite_1(mannitol) 40.3 ± 1.6 34.1 ± 4.1 30.1 ± 2.4 Metabolite_2 (sucralose)32.9 ± 4.6 30.2 ± 2.8 25.1 ± 1.4 Intermediate metabolite 70.3 ± 2.8 56.5± 1.7 40.5 ± 2.3 (C6-mannitol with protecting groups, Formula C)

The CYP 2E1 inhibition ratios of the test compound detected in the humanliver microsomes are shown in Table 1. From the results, test compounds,including the pro-drug (compound F) and its metabolites i.e. mannitol,sucralose and C6-mannitol with protecting group (Formula C), have beendemonstrated to be effective as P450 2E1 inhibitors, among which 4 μg/mLintermediate metabolite of pro-drug (i.e. C6-mannitol with protectinggroups, Formula C) showed the best inhibition effect (70.3±2.8%).

Example 5: Assays of Liver Injuries Induced by Acetaminophen (APAP) andCCl₄

5.1 Materials and Methods

5.1.1 Reagents

All organic solvents are HPLC grade and are purchased from Tedia(Fairfield, Ohio, USA). APAP is purchased from Sigma (St. Louis, Mo.USA), galactose injectable solution is manufactured by SouthernPhotochemical Co. and is prepared by dissolving 400 g of galactose(Sigma) in 1 L of buffer solution containing isotonic salts forinjections.

5.1.2 Animals

Male SD (Sprague-Dawley) rats weighing 175-280 g were purchased from theNational Laboratory Animal Center (NLAC), Taiwan. The study wasconducted in accordance with the Guidelines for Conducting AnimalStudies of the National Health Research Institute and all rats wereplaced in the air/humidity controlled environment under the 12 hours ofday/12 hours of night cycle and with unlimited water and food supply.During the course of the study, the weights of rats were monitoredcontinuously with normal water supply.

5.1.3 Treatments

5.1.3.1 Liver Injuries Induced by APAP

Mannitol and sucralose were used to perform the animal test (rat) inview of liver injuries induced by APAP.

In the normal control (Group 1), animals were not fed with APAP. In thecontrol group of APAP-induced liver injuries (Group 2), animals were fedwith a single dose of APAP in the amount of 2,000 mg per kilogram ofbody weight to induce hepatotoxicity. In the positive control group oftreatment with NAC (Group 3), animals were fed with a single dose ofAPAP in the amount of 2,000 mg per kilogram of body weight to inducehepatotoxicity, and 4 hours later, a 24-hour treatment period by tubefeeding was started, including first administration of 140 mg of NAC(per kilogram of body weight) and later administration of 70 mg of NAC(per kilogram of body weight) every 4 hours for five times. In theexperimental group (Group 4), animals were fed with a single dose ofAPAP in the amount of 2,000 mg per kilogram of body weight to inducehepatotoxicity, and 4 hours later, a 24-hour treatment period by tubefeeding was started, including six dosing with the ingredients of thepresent invention every 4 hours, as follows:

-   -   (a) (Group 4.1): administration of mannitol at a dose less than        or equivalent to 100 mg per person every 4 hours for 24 hours,    -   (b) (Group 4.2): administration of double dose of mannitol as in        Group 4.1 every 4 hours for 24 hours,    -   (c) (Group 4.3): administration of sucralose at a dose less than        or equivalent to 100 mg per person every 4 hours for 24 hours,    -   (d) (Group 4.4): administration of double dose of sucralose of        Group 4.3 every 4 hours for 24 hours,    -   (e) (Group 4.5): administration of a combination of 0.5 times        the dose of mannitol as in Group 4.1 and 0.5 times the dose of        sucralose as in Group 4.3 per kilogram of body weight every 4        hours for 24 hours,    -   (f) (Group 4.6): administration of a combination of the dose of        mannitol as in Group 4.1 and the dose of sucralose as in Group        4.3 every 4 hours for 24 hours,    -   (g) (Group 4.7): administration of a combination of 1.5 times        the dose of mannitol as in Group 4.1 and 1.5 times the dose of        sucralose as in Group 4.3 every 4 hours for 24 hours,    -   (h) (Group 4.8): administration of a combination of double dose        of mannitol as in Group 4.1 and double dose of sucralose as in        Group 4.3 every 4 hours for 24 hours, and    -   (i) (Group 4.9): first administration of 140 mg of NAC per        kilogram of body weight and later administration of a        combination of 70 mg of NAC plus double dose of mannitol as in        Group 4.1 and double dose of sucralose as in Group 4.3 every 4        hours for five times.

After the 24-hour treatment period, blood was collected from the tailartery of the rats for AST/SLT assays. Subsequently, rats were subjectedto GSP tests. Finally, rats were sacrificed and histological analysiswas performed.

5.1.3.2 Liver Injuries Induced by CCl₄

Mannitol and sucralose were chosen from the active ingredients asdescribed herein to perform the animal test (mice) in view of liverinjuries induced by CCl₄.

In the normal control, animals were administered with normal saline byintraperitoneal injection. In the control group of CCl₄ induced liverinjuries, animals were intraperitoneally injected with 10 ml/kg CCl₄(40% in corn oil) to induce hepatotoxicity. In the experimental group,animals were intraperitoneally injected with 10 ml/kg CCl₄ (40% in cornoil) to induce hepatotoxicity, and 4 hours later, different ingredientsof the present invention were administered by tube feeding. Blood wascollected from the mice before administration with the ingredients ofthe present invention or at 24 hours after administration with theingredients of the present invention for AST/ALT assays. Finally,animals were sacrificed at day 2 and blood were collected for AST/ALTassay and histological analysis was performed.

On the other hand, other experimental groups of mice were fed with theingredients of the present invention for 12 weeks and the mice weresubjected to GSP tests.

5.1.4 Blood Samples

After completion of the treatments, rats were sacrificed under etheranesthesia, and blood was collected from the tail artery of the rats andplaced in a test tube containing EDTA. The plasma was centrifuged at13,000 at 4° C. for 15 minutes and the isolated plasma was transferredto Eppendorf tubes in aliquots and stored at −80° C.

5.1.5 Biochemical Analysis

Liver damage is quantified by measuring plasma AST and ALT activity. ASTand ALT are common indicators of hepatotoxicity and are measured byusing the Synchron LXi 725 system (Beckman Instruments, U.S.).

5.1.6 Optic Microscope

Following scarification of the rats, histological analysis wasperformed. Liver samples were fixed with 10% phosphate-bufferedformalin, dehydrated an embedded in paraffin, Sections were prepared in5 μm thickness and then stained with hematoxylin and eosin and subjectedto Periodic acid Schiff stain (PAS). The stained sections were observedunder the optic microscope.

5.1.7 Quantitative Tests of Liver Function

After the study was completed, all rats were subjected to GSP test. Ratswere i.v. injected with 0.4 g/ml BW galactose solution 0.5 g/kg within30 seconds and one blood sample was collected at 5, 10, 15, 30, 45 and60 minutes post injection from the tail vein. Colorimetric galactosedehydrogenase is used to quantify the concentration of galactose and thetest concentration ranges from 50 to 1,000 μg/ml. The within-dayvariation of each concentration is calculated using standard deviationand coefficient of variation (CV) and the maximum allowable coefficientof variation is 10% CV, whereas day-to-day variation is examined bycomparing the slope and intercept of calibration curves. The GSP is theblood galactose concentration obtained 60 seconds after stopping the30-second injection.

5.1.8 Statistical Analysis

All data are represented in mean±standard deviation (SD) and the resultsare calculated using ANOVA to determine the significance. StatisticalPackage of the Social Science program (Version 13, SPSS Inc.) is usedfor calculations followed by post hoc test to examine the leastsignificant difference for multiple comparisons so as to confirm thesignificant differences between groups and the average differencebetween groups was significant p<0.05.

5.2 Results

5.2.1 Mannitol and Sucralose and Other Ingredients are Effective inTreating Liver Injuries Induced by APAP

The results are shown in Table 2.

TABLE 2 Liver function GSP AST ALT Total Survival parameters (mg/L)(IU/L) (IU/L) HAI score (Day 14, n/n) Group 1: Normal 220 ± 24   186 ±16   65 ± 16  0.0 ± 0.0   3/3 control (NC, n = 6) Group 2: APAP 1017 ±170   1151 ± 310   746 ± 143  8.6 ± 0.5    2/12 control (2,000 mg/kg, n= 12) Group 3: NAC 393 ± 68*** 428 ± 74*** 221 ± 69*** 4.2 ± 0.8*** 3/6(140 mg/kg of NAC followed by 5 × 70 mg/kg NAC at 4 h intervals, n = 6)Group 4.1 (n = 3)  565 ± 177*** 455 ± 78*** 209 ± 16*** 4.0 ± 0.0*** 1/3(Mannitol at a dose less than or equivalent to 100 mg per person) × 6Group 4.2 (n = 3) 354 ± 56*** 300 ± 40*** 166 ± 15*** 4.0 ± 1.0*** 3/3(Double dose of Group 4.1 (mannitol)) × 6 Group 4.3 (n = 3) 332 ± 42***331 ± 41*** 154 ± 49*** 4.0 ± 1.0*** 3/3 (Sucralose at a dose less thanor equivalent to 100 mg per person) × 6 Group 4.4 (n = 3) 309 ± 54***277 ± 78*** 136 ± 48*** 3.0 ± 1.0*** 3/3 (Double dose of Group 4.3(sucralose)) × 6 Group 4.5 (n = 3) 332 ± 61***  3 0 ± 81*** 149 ± 19***2.0 ± 1.0*** 3/3 (0.5 times the dose of Group 4.1 (mannitol) + 0.5 timesthe dose of Group 4.3 (sucralose)) × 6 Group 4.6 (n = 3) 271 ± 52*** 193± 34***  81 ± 18*** 1.5 ± 1.0*** 6/6 (the dose of Group 4.1 (mannitol) +the dose of Group 4.3 (sucralose)) × 6 Group 4.7 (n= 3) 265 ± 53*** 203± 24***  83 ± 25*** 1.0 ± 1.0*** 3/3 (1.5 times the dose of Group 4.1(mannitol) + 1.5 times the dose of Group 4.3 (sucralose)) × 6 Group 4.8(n = 3) 227 ± 25*** 159 ± 21***  69 ± 10*** 0.5 ± 0.5*** 6/6 (doubledose of Group 4.1 (mannitol) + double dose of Group 4.3 (sucralose)) × 6Group 4.9 (n = 3) 233 ± 41*** 171 ± 25*** 58 ± 9*** 0.3 ± 0.5*** 6/6 140mg/kg NAC + 5 × (70 mg NAC + double dose of Group 4.1 (mannitol + doubledose of Group 4.3 (sucralose)) Group 5 (n = 6) 280 ± 98*** 247 ± 43*** 66 ± 18*** 2.8 ± 1.0*** 6/6 (Aerosil 200 at a dose less than orequivalent to 100 mg per person) Group 6 (n = 6) 294 ± 30*** 248 ± 37*** 81 ± 27*** 2.7 ± 1.2*** 6/6 (Sodium starch glycolate at a dose lessthan or equivalent to 100 mg per person) Group 7 (n = 6) 372 ± 90*** 323± 40*** 175 ± 61*** 2.8 ± 1.5*** 6/6 (Crospovidone at a dose less thanor equivalent to 100 mg per person) Group 8 (n = 6) 259 ± 36*** 217 ±28***  72 ± 21*** 2.2 ± 1.0*** 6/6 (Microcrystalline cellulose at a doseless than or equivalent to 100 mg per person) Group 9 (n = 6) 287 ±38*** 220 ± 53***  71 ± 26*** 2.5 ± 1.0*** 6/6 (Povidone K-30 at a doseless than or equivalent to 100 mg per person) *p < 0.05, **p < 0.01,***p < 0.005: comparison of the experimental groups with APAP control

The results show that liver injuries has occurred in the APAPhepatotoxicity group. In contrast, such liver injuries and survival ratecan be improved by use of mannitol and/or sucralose, in a dose dependentmanner. Especially, a combination of mannitol and sucralose achieves asynergistic effect; the results are similar to those of normal controland even better than the positive control of standard treatment withNAC. In addition, other ingredients including Aerosil 200, Sodium starchglycolate, Crospovidone, Microcrystalline cellulose and Povidone K-30are found effective in treating the liver injuries, also better than thepositive control of standard treatment with NAC.

The improved results are also reflected in the corresponding livertissues.

FIG. 4 shows the results of the histological analysis. The liver tissuesections from the rats in the APAP hepatotoxicity group showed thathepatocytes surrounding the central vein are broken with visiblevacuolization and reduced number of nucleuses, some hepatocytes evenshowed the signs of necrosis and liver damage is more severe whencompared with the hepatocytes from rats in the normal control group(FIG. 4B). On the contrary, liver structure of rats in the control groupare normal, the hepatocytes are intact and arranged in order with novacuolization (FIG. 4A). As for the liver sections from the experimentalgroups with treatment by mannitol and/or sucralose, the hepatocytes arerelatively intact with visible nucleus and less vacuolization (FIG. 4D,E, F, G, H). Especially, a combination of mannitol and sucraloseachieves the best protective effect (FIG. 4G); the results are evenbetter than the positive control of standard treatment with NAC (FIG.4C).

5.2.2 Mannitol is Effective in Treating Liver Injuries Induced by CCl₄

The results are shown in Table 3.

TABLE 3 Liver function parameters GSP AST ALT Total HAI Groups (mg/L)(IU/L) (IU/L) score Normal control 315 ± 48   88 ± 20 57 ± 17   0.0 ±0.0   (n = 10) CCl₄ control group 914 ± 205*** 815 ± 216*** 770 ± 274***6.2 ± 2.1*** (n = 10) Dose of kaempfrol less 456 ± 101*** 198 ± 105***128 ± 40***  4.3 ± 13*   than or equivalent to 100 mg per person (n =10) Dose of 312 ± 140*** 144 ± 49*** 95 ± 36*** 1.7 ± 0.9***epigallocetechin-3-gallate less than or equivalent to 100 mg per person(n = 10) Dose of quercetin less 286 ± 70***  115 ± 40*** 93 ± 26*** 1.1± 0.7*** than or equivalent to 100 mg per person (n = 10) Dose ofmannitol less 290 ± 78***  91 ± 28*** 77 ± 22*** 0.8 ± 0.5*** than orequivalent to 100 mg per person (n = 10) Statistic analysis: Anova andLSD tests. ***p < 0.005, **p < 0.01, *p < 0.05, comparison of theexperimental groups with CCl₄ control group.

The results show that liver injuries has occurred in the CCl₄ controlgroup. In contrast, such liver injuries can be improved by use ofmannitol.

Example 6: Assays of Fatty Liver

6.1 Materials and Methods

6.1.1 Cell Lines and Cell Culture Media

The activity of the various ingredients as described herein, includingmannitol and sucralose and others, in reduction of fat content wasanalyzed by using human hepatoma cell line Hep G2.

Dulbecco's Modified Eagle's Medium (DMEM) was used to prepare DMEMculture Nos. A-F listed in Table 4 for carrying out subsequentexperiments.

TABLE 4 Preparations of DMEM culture media Nos. A-F DMEM culturesPreparation methods No. A DMEM was dissolved in 1,400 mL of water withstirring, and then 2 g of 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonicacid (HEPES) was added to form a solution, to which a sodium bicarbonatesolution (4 g of sodium bicarbonate powder dissolved in 400 mL of waterby stirring) was added, and the volume was made up to 2,000 mL withwater. The pH of the resulting solution was adjusted to 7.3 ± 0.05 byadding 5N HCl. After being filtered through a 0.2 μm sterile membrane,the final solution was dispensed into sterile serum vials and stored at4° C. No. B 50 mL of deactivated fetal bovine serum (FBS), 5 mL ofsodium pyruvate (100 mM), 5 mL of penicillin (100 U/mL) and streptomycin(100 U/mL), and 5 mL of MEM non-essential amino acid solution(100X) wereadded into 450 mL of DMEM culture No. A. No. C 5 mL of sodium pyruvate(100 mM), 5 mL of penicillin (100 U/mL) and streptomycin (100 U/mL), and5 mL of MEM non-essential amino acid solution(100X) were added into 450mL of DMEM culture No. A. No. D DMEM culture No. B was added into theoleate/albumin complex. The oleate/albumin complex was preparedaccording to the method presented by Van Harken et al. in 1969 (J BiolChem. 1969 May 10; 244(9): 2278-85). The method included taking 25 mL ofDMEM culture No. A, into which 5 g of bovine serum albumin (BSA) wasadded, and then 5N sodium hydroxide solution was added to adjust the pHto 7.4 to form a mixture. The mixture was then placed in an ice bath at0° C. to form the BSA solution. The oleic acid was dissolved in 50 ml ofalcohol (95%) and then titrated to the phenolphthalein titrationendpoint with 1N sodium hydroxide solution. The alcohol was blown awayby flowing helium. The resulting sodium oleate was dissolved in DMEMculture No. A at 37° C. to form a sodium oleate solution. At last, theBSA solution was added dropwise into the sodium oleate solution withstirring to form the oleate/albumin complex solution. No. E Variousamounts silymarin were dissolved in DMEM culture No. C. No. F Variousamounts of the test compounds of the present invention were dissolved inDMEM culture No. C.

The DMEM cultures Nos. A-F were preserved at 2-8° C., and warmed up in awater bath at 37° C. before the experiments.

6.1.2 Cell Counts and Survivability Test

Dead cells would take up 0.4% trypan blue and then had a color; whereaslive cells exclude certain dyes due to the intact cell membranes and hada clear color. 100 μl of cell suspension and equal volume of 0.4% trypanblue were mixed uniformly to form a mixture. Some of the mixture (about20 μl) was added into the groove above the chamber of the hemocytometer,which was then covered with a cover slip for observing under the opticalmicroscope. Live cells were not stained, and dead cells were blue.

6.1.3 Oleic Acid-Induced Formation of Fatty Liver Cells from HepG2 CellLines

HepG2 cell lines (15×10⁶ cells) were cultured in DMEM culture No. B,incubated in an incubator with 5% CO₂ at 37° C. for 24 hours, culturedin DMEM culture No. C (serum-free medium) for 24 hours, and finallycultured in DMEM culture No. D (containing oleate/albumin complex) foranother 48 hours to induce HepG2 cell lines to form fatty liver cells.

6.1.4 Treatments for Each Group of Fatty Liver Cells

HepG2 cell lines were divided into six groups, including: (1) Blank: notreatment; (2) DMSO group: cells from Blank were treated with dimethylsulfoxide (DMSO); (3) Control: induction with oleic acid to form fattyliver cells; (4) Vehicle group: fatty liver cells formed by inductionwith oleic acid were treated with DMSO; (5) Positive control: fattyliver cells were treated with silymarin, and (6) Test Group: fatty livercells were treated with various compounds of the present Invention.

6.1.5 Determination of triglyceride (TG) in cells

After incubation for 72 hours, the treated cells from each group weresuccessively washed twice in PBS, and then incubated with 0.5 ml oftrypsin/EDTA for 3 minutes. Afterwards, the cells were scraped with 2 mlof PBS and then transferred to the centrifuge tube to be shattered byultrasonic. A volume of 20 μl cell extracts was taken to determine thecontent of protein. TG determination was performed using commerciallyavailable combination of agents (Randox). The TG content obtained abovewas divided by the protein content to get a ratio, which represented therelative content of TG in cells.

6.1.6 Animals for Experiments

B6 mice recommended in the specification “Method for evaluating theliver protection and health care efficacies of health food” announced bythe Department of Health of Taiwan were chosen for animal testing. Morethan four mice were used in each group of the pre-test, while more thantwelve mice were used in each group of the confirmatory test. Male micebred at 23±2° C. in an animal room with 55±15% relative humidity undernormal light/dark cycle (7:00 AM-7:00 PM lights on/7:00 PM-7:00 AMlights off) and weighing 18-23 g were purchased from BioLASCO (Taipei)and housed at Laboratory Animal Center in National Defense MedicalCenter. The animal test was carried out according to the guideline foranimal experiment of National Health Research Institutes. Mice were fedwith normal feed at 3-5 g/day and unlimited supply of water for 1-2weeks and investigated for health condition. The weight of mice wasrecorded once a week.

6.1.7 Animal Grouping

The tested animals were grouped randomly into Blank, High Fat Dietcontrol (HFD), Positive Control (PS), and Test group. The animals ofBlank were fed with normal feed. The animals of HFD were fed with highfat feed. The animals of PS were fed with high fat feed, andadditionally fed with silymarin (5 mg/kg/day) by a tube. The animals ofTest group were fed with high fat feed, and additionally fed with testcompounds by a tube.

6.1.8 Test Methods

The animals of Blank were fed casually with normal feed for 12 weeks,while the animals of HFD, PS, and Test group were fed casually with highfat feed for 12 weeks. After 8 weeks of feeding, the animals of Blankand HFD were fed with deionized water by a tube once a day; the animalsof PS were fed with silymarin by a tube once a day; and the animals ofTest Group were fed with test compounds by a tube once a day for aduration of 4 or 8 weeks.

Before testing and in the eighth, twelfth, and sixteenth week aftertesting, blood was collected from the cheek or the heart. At the end oftesting, all mice were weighted and then sacrificed, and blood wascollected from the cheek or the heart simultaneously. The bloodspecimens of mice rested at room temperature for one hour to clot, andthen the serum was separated by centrifugation in a refrigerationcentrifuge at 15,700×g at 4° C. for 5 minutes. Afterwards, biochemicalindices of liver function, including aspartate transaminase (AST),alanine aminotransferase (ALT), triglyceride (TG), total cholesterol(TCHO/TC), low-density lipoprotein cholesterol (LDL-C), and high-densitylipoprotein cholesterol (HDL-C), were detected by the automatic bloodbiochemistry analyzer.

In addition, abdominal fat and liver specimens were taken from theabdomens of sacrificed mice and weighted to compare the weight of fatand liver and obtain the ratio of liver weight to body weight. Twotissue blocks with a volume of approximately 1 cm³ were cut from thelargest right lobe of liver, fixed in 10% neutral formalin solution, andthen embedded with paraffin for sectioning. The cut sections proceededwith H&E staining for histopathological observation. Moreover, the restof the liver was frozen for preservation and detection of the contentsof triglyceride and total cholesterol in the liver. Furthermore, theliver function of animals of each group were analyzed by GalactoseSingle Point Method, which was recognized and recommended forquantification of remaining liver function in clinical use by U.S. FDAand Ministry of Health and Welfare, Taiwan. At the end of the tests, 0.5g of galactose (G.S.P.® 0.4 g/mL) per kg of animal was administered viaintravenous. One hour after the administration, about 0.5 ml of wholeblood was taken by using a filter paper to evaluate liver function ofmice. The higher the value of GSP was, the worse the remaining liverfunction would be. (FDA: “Guidance for Industry: Pharmacokinetics inPatients with Impaired. Hepatic Function-Study Design, Data Analysis andImpact on Dosing and. Labeling. 2003.

6.1.9 Histopathological Tissue Sectioning:

At the end of the test, all mice were sacrificed. One tissue block witha volume of approximately 1 cm³ was cut from the largest right lobe ofliver, fixed in 10% neutral formalin, and then dehydrated and hyalinizedin various concentrations of ethanol (30{grave over ( )}50{grave over( )}70{grave over ( )}95{grave over ( )}99.5%) and xylene. Afterwards,xylene was replaced with hot paraffin solution. At last, the tissue wasembedded with paraffin solution. The finished paraffin specimen was cutinto 5 μm-thickness paraffin sections by the microtome. The sectionswere pasted on clean slides, dried at 37° C., and then stained by H&Estaining.

6.1.10 Hematoxylin and Eosin Staining (H&E)

Liver tissue sections were deparaffinized in xylene for 30 minutes, andthen successively rehydrated twice in 99.5%, 95%, 70%, 50%, and 30%aqueous ethanol for 30 minutes respectively. After being soaked indistilled water for 10 minutes, the sections could be stained. Thesections were first immersed in hematoxylin for 30 seconds to stain cellnuclei, then washed with distilled water for a few minutes, stained witheosin for 2-5 minutes, and washed with distilled water for a few minutesagain. After staining process was finished, the sections were dehydratedsuccessively in 50%, 70%, 95%, and 100% aqueous ethanol twice for 30seconds respectively, hyalinized twice in xylene, and finally sealed andstored with mounting media.

6.1.11 Histopathological Observation

In order to observe the changes of lesion, fat accumulation, necrosis,or fibrosis in liver cells when there was an ongoing liver damage, livertissues were H&E stained to evaluate the degree of liver fataccumulation. All the histopathological sections were cut from the sameposition on the largest right lobe of liver for eliminating bias insubjective observation, and then subject to pathological staining. Asfor the assessment of semi-quantitative analysis in pathology, it had tobe confirmed by a physician or a veterinary pathologist who conducted adouble-blind analysis to score (NAS score) and compare all the sectionswithout knowing the test design. At last, the differential analysis ofeach group was performed by statistical methods.

6.1.12 Analysis of Liver Antioxidant Capacity

About 0.1 g of liver tissue was taken from the sacrificed animal andhomogenized by centrifuge with a biomasher for 10 minutes. A 9-foldweight (w/w) of buffer (pH 7.4, 50 mmol/L Tris-HCl, 180 mmol/L KCl) wasadded to the homogenized tissue, which was then mixed well by a Vortexmixer for use. The resulting homogenization solution samples of livertissue was used to analyze the various members of liver antioxidantsystems, including glutathione peroxidase (GPx), glutathione (GSH),glutathione reductase (Grd), and superoxide dismutase (SOD). Methods ofrelated analysis can be found in the known literatures, for example, thedraft of “Method for evaluating the liver protection and health careefficacies of health food” announced by the Ministry of Health andWelfare, Taiwan.

6.1.13 Statistical Analysis

All data were expressed as means±standard deviation (SD). Statisticallysignificant difference of the test results was determined by calculationof one-way ANOVA using Statistical Package of the Social Scienceprogram, Version 13, SPSS Inc. Thereafter, multiple comparisons werecarried out by using least significant difference method in post hoctest to confirm the significant difference between groups. The averagedifference between groups is judged to be significant when p<0.05.

6.2 Results

6.2.1 Cell Experiments

In cell experiments, the results of TG content reduction in HepG2 cellsdetermined in Positive Control (silymarin) were listed in Table 5.

TABLE 5 Efficacy of silymarin in reduction of TG content in HepG2 fatcells of Positive Control Silymarin concentration TG content in cellsReduction rate of (μM) (μg/mg protein) TG (%) 0 (Control) 59.43 ± 4.60 —1.0 44.17 ± 2.41 29 ± 8  5.0  44.59 ± 11.53 28 ± 10 1.0 26.38 ± 9.12 63± 11 100 20.48 ± 4.76 78 ± 5 

The results of TG content reduction in HepG2 fat cells determined usingconstant concentration of test compounds were shown in Table 6. It canbe seen from the results that the test compounds exhibited differentdegrees of TG content reduction effects in fatty liver cells formed frominduced HepG2 cells under the condition of constant test concentration,as compared with Control. The equation for calculating reduction rate(%) of TG was as follows: [1−(TG content of Test Group−TG content ofBlank)/(TG content of Oleic acid induction Group−TG content ofBlank)]×100%.

TABLE 6 TG content in fatty liver cells reduced by test compounds Testedsubstances (1.0 μM) TG reduction rate (%) Silymarin Control 35.33 ± 1.96Puerarin 49.91 ± 7.73 Phloridzin 42.35 ± 6.05 Daidzein  42.3 ± 5.34Sodium lauryl sulfate 38.73 ± 4.65 Poncirin 38.12 ± 7.22 Sinensetin36.97 ± 4.84 (−)-Epigallocatechin 36.78 ± 6.67 Kaempferol 36.51 ± 4.78Isovitexin 35.93 ± 3.35 Ursolic Acid 35.86 ± 8.92 Eriodictyol 35.11 ±0.87 (+)-Limonene  35.02 ± 10.04 Hesperidin 34.81 ± 5.25 Ergosterol34.19 ± 3.69 β-myrcene  33.97 ± 11.22 (−)-Epicatechin-3-gallate  32.7 ±4.33 Hyperoside 30.51 ± 2.8  Silybin 30.26 ± 3.24 (+)-Catechin 29.57 ±4.02 Formononetin 29.55 ± 1.44 Myristic acid ethyl ester 28.88 ± 3.91Galangin 28.11 ± 8.62 Suralose 26.68 ± 2.93 Eicosapentaenoic acid (EPA)26.15 ± 6.14 Morin  25.84 ± 10.65 Mannitol 22.35 ± 5.74 Sciadopitysin21.83 ± 5.04 Wongonin 20.78 ± 1.12 Didymin  20.37 ± 12.69 Gossypin 20.25± 4.63 Sorbitol 20.06 ± 2.57 Luteolin-7-glucoside 19.33 ± 4.59 PovidoneK-30 18.93 ± 5.13 Protocatechuic acid 18.57 ± 7.6  (+)-Taxifolin 17.91 ±8.35 Saccharin 17.53 ± 6.96 Umbelliferone  17.4 ± 2.57 Glycerin 16.23 ±4.25 Hesperitin 16.08 ± 5.55 Nordihydroguaiaretic acid 15.92 ± 2.3 Trans-Cinnamic Acid 15.85 ± 0.82 Sodium benzoate 14.35 ± 4.86 Oxide red13.59 ± 2.08 Neohesperidin 13.29 ± 7.21 Naringin 12.69 ± 3.72 Diosmin11.86 ± 3.73 (−)-Epicatechin 10.76 ± 8.92 Glycyrrhizin 10.55 ± 7.4 Linarin  9.24 ± 12.34 Baicalin  9.21 ± 6.21 Quercitrin  9.15 ± 9.24Xylitol  7.36 ± 6.34 Baicalein  7.09 ± 10.88 Luteolin  6.95 ± 15.23Swertiamarin  6.72 ± 11.04 Butylated hydroxyanisole 6.21 ± 3.8 Sodiumcyclamate  4.77 ± 4.49 Menthol 66.24 ± 1.87 Citric acid  2.55 ± 4.43Lemon oil  0.56 ± 1.07 Pregelatinized starch  7.18 ± 13.41 Sorbic acid 2.03 ± 1.96

TABLE 6-1 A portion of test compounds from Table 6 that reduced TGcontent in fatty liver cells Tested substances (1.0 uM) TG reductionrate (%) Puerarin 49.91 ± 7.73 Phloridzin 42.35 ± 6.05 Daidzein  42.3 ±5.34 Sinensetin 36.97 ± 4.84 (−)-Epigallocatechin 36.78 ± 6.67Kaempferol 36.51 ± 4.78 Ursolic Acid 35.86 ± 8.92 Silymarin of Control35.33 ± 1.96 (+)-Limonene  35.02 ± 10.04 Hesperidin 34.81 ± 5.25(−)-Epicatechin-3-gallate  32.7 ± 4.33 Silybin 30.26 ± 3.24 Formononetin29.55 ± 1.44 Myristic acid ethyl ester 28.88 ± 3.91 Eicosapentaenoicacid (EPA) 26.15 ± 6.14 Wongonin 20.78 ± 1.12 Povidone K-30 18.93 ± 5.13Protocatechuic acid 18.57 ± 7.6  Umbelliferone  17.4 ± 2.57 Hesperitin16.08 ± 5.55 Nordihydroguaiaretic acid 15.92 ± 2.3  Neohesperidin 13.29± 7.21 Naringin 12.69 ± 3.72 (−)-Epicatechin 10.76 ± 8.92 Glycyrrhizin10.55 ± 7.4  Baicalin  9.21 ± 6.21 Quercitrin  9.15 ± 9.24 Baicalein 7.09 ± 10.88

TABLE 6-2 A portion of test compounds (Bioflavonoids) from Table 6 thatreduced TG content in fatty liver cells Tested substances (1.0 uM) TGreduction rate (%) Poncirin 38.12 ± 7.22 Isovitexin 35.93 ± 3.35Eriodictyol 35.11 ± 0.87 Ergosterol 34.19 ± 3.69 β-myrcene 33.97 ± 11.22Hyperoside 30.51 ± 2.8 (+)-Catechin 29.57 ± 4.02 Galangin 28.11 ± 8.62Morin 25.84 ± 10.65 Sciadopitysin 21.83 ± 5.04 Didymin 20.37 ± 12.69Gossypin 20.25 ± 4.63 Luteolin-7-glucoside 19.33 ± 4.59 (+)-Taxifolin17.91 ± 8.35 Trans-Cinnamic Acid 15.85 ± 0.82 Diosmin 11.86 ± 3.73Linarin  9.24 ± 12.34 Xylitol  7.36 ± 6.34 Luteolin  6.95 ± 15.23Swertiamarin  6.72 ± 11.04

TABLE 6-3 A portion of test compounds (excipients) from Table 6 thatreduced TG content in fatty liver cells Tested substances (1.0 uM) TGreduction rate (%) Sodium lauryl sulfate 38.73 ± 4.65 Sucralose 26.68 ±2.93 Mannitol 22.35 ± 5.74 Sorbitol 20.06 ± 2.57 Saccharin 17.53 ± 6.96Glycerin 16.23 ± 4.25 Sodium benzoate 14.35 ± 4.86 Oxide red 13.59 ±2.08 Butylated hydroxyanisole  6.21 ± 3.8 Sodium cyclamate  4.77 ± 4.49Menthol 66.24 ± 1.87 Citric acid  2.55 ± 4.43 Lemon oil  0.56 ± 1.07Pregelatinized starch  7.18 ± 13.41 Sorbic acid  2.03 ± 1.96

6.2.2 Animal Experiments

In the animal experiments, all the animals were treated to induce fattyliver, except the animals of Blank that were fed with normal feed. Aftereight weeks, the animals of each group were given different treatmentfor four or eight weeks in addition to the original feed. The animals ofBlank and HFD were fed with deionized water; the animals of PS were fedwith silymarin; and the animals of Test Group were fed with differenttest compounds, including puerarin, phloridzin, eriodictyol, sucralose,mannitol, saccharin, hesperitin, menthol, and combinations thereof

6.2.2.1 the Effects on Body Weight, Liver Weight, and Weight of Body Fatof Animals and Safety Evaluation of Test Compounds

From the results of animal experiments, the liver weight, weight of bodyfat, and increase of body weight of animals of each group were listed inTable 7-1 and 7-2.

TABLE 7-1 The analysis results of liver weight and weight of body fatdue to test compounds Abdominal fat weight Liver weight Unit Items g gBlank (n = 13) 0.6 ± 0.2*** 1.6 ± 0.2 0.6 HFD (n = 12) 2.8 ± 0.4 1.6 ±0.4 2.8 Positive Control Silymarin 5.0 mg/kg (n = 6) 2.0 ± 0.4*** 1.2 ±0.3*** Silymarin 1.5 mg/kg (n = 6) 2.3 ± 0.5* 1.5 ± 0.1 Single testcompound Phloridzin 2.5 mg/kg (n = 6) 2.3 ± 0.6* 1.3 ± 0.1* Eriodictyol2.5 mg/kg (n = 6) 2.7 ± 0.6 1.3 ± 0.1** Sucralose 7.5 mg/kg (n = 6) 2.4± 0.3 1.4 ± 0.1 Sucralose 1.5 mg/kg (n = 6) 2.1 ± 0.6** 1.5 ± 0.2Menthol 1.5 mg/kg (n = 6) 2.3 ± 0.6* 1.6 ± 0.2 Mannitol 7.5 mg/kg (n =6) 2.4 ± 0.3 1.4 ± 0.1 Mannitol 4.5 mg/kg (n = 6) 2.7 ± 0.3 1.4 ± 0.2Mannitol 1.5 mg/kg (n = 6) 2.0 ± 0.3*** 1.4 ± 0.2 Saccharin 1.5 mg/kg (n= 3) 2.3 ± 0.5 1.5 ± 0.1 Puerarin 2.5 mg/kg (n = 6) 2.8 ± 0.3 1.4 ± 0.2Hesperitin 2.5 mg/kg (n = 6) 3.0 ± 0.5 1.5 ± 0.1 Combinations of twotest compounds Saccharin + Mannitol 2.7 ± 0.4 1.4 ± 0.2 2.7 1.5 mg/kg +1.5 mg/kg (n = 6) Menthol + Mannitol 3.0 ± 0.5 1.6 ± 0.3 3.0 4.5 mg/kg +4.5 mg/kg (n = 6) Menthol + Mannitol 2.3 ± 0.6 1.5 ± 0.3 2.3 1.5 mg/kg +1.5 mg/kg (n = 6) Combinations of three test compounds Menthol +Mannitol + Eriodictyol 2.6 ± 0.6 1.4 ± 0.2 2.6 .5 mg/kg + .5 mg/kg + .8mg/kg(n = 6) Data were expressed as means ± SD. Statistical differenceresulted from ANOVA and LSD was denoted by words. *p < 0.05, **p < 0.01,***p < 0.005, as compared with HFD. Hesperitin Puerarin EriodictyolPhloridzin Mannitol Menthol Sucralose Saccharin TG: triglyceride TC:total cholesterol

TABLE 7-2 The analysis results of increase of body weight due to testcompounds Increase of body weight Unit Items g Blank (n = 13) 15.6 ± 7.9HFD (n = 12) 14.0 ± 8.4 Positive Control Silymarin 5.0 mg/kg (n = 6)10.2 ± 12.7 Silymarin 1.5 mg/kg (n = 6) 10.9 ± 4.3 Single test compoundPhloridzin 2.5 mg/kg (n = 6) 13.7 ± 10.7 Eriodictyol 2.5 mg/kg (n = 6) 8.3 ± 6.7 Sucralose 7.5 mg/kg (n = 6)  8.3 ± 5.4 Sucralose 1.5 mg/kg (n= 6) 17.0 ± 5.6 Menthol 1.5 mg/kg (n = 6) 19.6 ± 5.0 Mannitol 7.5 mg/kg(n = 6) 10.3 ± 8.5 Mannitol 4.5 mg/kg (n = 6) 11.1 ± 7.7 Mannitol 1.5mg/kg (n = 6) 10.9 ± 7.4 Saccharin 1.5 mg/kg (n = 3) 27.7 ± 12.7**Puerarin 2.5 mg/kg (n = 6) 21.7 ± 3.1* Hesperitin 2.5 mg/kg (n = 6) 14.5± 8.3 Combinations of two test compounds Saccharin + Mannitol 16.6 ± 6.41.5 mg/kg + 1.5 mg/kg (n = 6) Menthol + Mannitol 15.6 ± 5.0 4.5 mg/kg +4.5 mg/kg (n = 6) Menthol + Mannitol 14.9 ± 6.3 1.5 mg/kg + 1.5 mg/kg (n= 6) Combinations of three test compounds Menthol + Mannitol +Eriodictyol 21.7 ± 3.9* .5 mg/kg + .5 mg/kg + .8 mg/kg (n = 6) Data wereexpressed as means ± SD. Statistical difference resulted from ANOVA andLSD was denoted by words. *p < 0.05, **p < 0.01, ***p < 0.005, ascompared with HFD. Hesperitin Puerarin Eriodictyol Phloridzin MannitolMenthol Sucralose Saccharin TG: triglyceride TC: total cholesterol

It was shown from the results that the weight of abdominal fat increasedin animals induced with fatty liver. Among the test compoundsadministered separately, mannitol, menthol, and sucralose could reducethe weight of abdominal fat in animals significantly.

In addition, no abnormal condition was observed in animals of Test Groupafter the test compounds were administered. No animal died during thetest. Occurrence of diseases or clinical symptoms caused by the testcompounds was not observed from necropsy studies of sacrificial animalsafter the tests. Therefore, the test compounds were safe.

6.2.2.2 the Test Compounds are Effective in Reducing Lipid in Liver

FIG. 5 showed the mice that were induced to exhibit fatty liver whoseliver cells near hepatic portal area (including the bile duct, portalvein, hepatic artery) were covered with many large vesicular fatdroplets and hepatocellular ballooning appeared, indicating that theanimal model of fatty liver was successfully established by induction.

The results of animal experiments showed that a plurality of testcompounds exhibited the effects of lipid reduction in animal liversafter administration for a period of 4 or 8 weeks. The results wereshown in Tables 8-1 and 8-2.

TABLE 8-1 Test compounds could reduce liver lipids in animals(administration period of 4 weeks) TG in liver TC in liver Unit Itemsmg/g liver mg/g liver Blank (n = 13) 25.0 ± 9.2***  2.5 ± 0.4*** HFD (n= 12) 132.0 ± 69.2   6.6 ± 3.5   Positive Control Silymarin 5.0 mg/kg (n= 6) 46.8 ± 14.4*** 3.0 ± 0.9*** Silymarin 1.5 mg/kg (n = 6) 69.9 ±32.3**  3.7 ± 0.4**  Single test compound Phloridzin 2.5 mg/kg (n = 6)48.9 ± 14.1*** 2.9 ± 0.5*** Eriodictyol 5.0 mg/kg (n = 6) 54.2 ± 15.0***3.0 ± 0.9*** Eriodictyol 2.5 mg/kg (n = 6) 43.1 ± 13.1*** 3.8 ± 1.1** Sucralose 7.5 mg/kg (n = 6) 56.8 ± 20.0*** 5.0 ± 0.9   Sucralose 1.5mg/kg (n = 6) 68.9 ± 37.5**  3.0 ± 0.9*** Menthol 1.5 mg/kg (n = 6) 87.3± 72.3*  4.4 ± 3.5*  Mannitol 7.5 mg/kg (n = 6) 53.8 ± 24.4*** 4.7 ±1.2   Mannitol 4.5 mg/kg (n = 6) 71.5 ± 45.5*** 7.2 ± 2.8   Mannitol 1.5mg/kg (n = 6) 61.8 ± 32.6*** 3.4 ± 0.6*** Saccharin 1.5 mg/kg (n = 3)84.0 ± 41.4   2.8 ± 1.5**  Puerarin 2.5 mg/kg (n = 6) 89.4 ± 49.1*  6.7± 2.7   Hesperitin 2.5 mg/kg (n = 6) 67.8 ± 16.6*** 3.7 ± 0.7** Combinations of two test compounds Saccharin + Mannitol 71.6 ± 32.0***8.5 ± 2.5   1.5 mg/kg + 1.5 mg/kg (n = 6) Menthol + Mannitol 54.3 ±11.8*** 4.5 mg/kg + 4.5 mg/kg (n = 6) Menthol + Mannitol 31.0 ± 11.2***6.9 ± 1.7   1.5 mg/kg + 1.5 mg/kg (n = 6) Menthol + Mannitol 96.6 ±77.4   5.9 ± 1.7   .5 mg/kg + .5 mg/kg (n = 6) Combinations of threetest compounds Menthol + Mannitol + Eriodictyol 83.1 ± 50.9*  6.0 ±2.3   .5 mg/kg + .5 mg/kg + .8 mg/kg (n = 6) Data were expressed asmeans ± SD. Statistical difference resulted from ANOVA and LSD wasdenoted by words. *p < 0.05, **p < 0.01, ***p < 0.005, as compared withHFD. Hesperitin Puerarin Eriodictyol Phloridzin Mannitol MentholSucralose Saccharin TG: triglyceride TC: total cholesterol

TABLE 8-2 Test compounds could reduce liver lipids in animals(administration period of 8 weeks) TG in liver TC in liver Unit Itemsmg/g liver mg/g liver Blank (n = 7) 22.6 ± 3.8***  3.8 ± 0.4*** HFD (n =8) 187.3 ± 91.2   12.1 ± 7.3   Combinations of two test compoundsSucralose + Mannitol 115.3 ± 36.2*   6.0 ± 3.0**  7.5 mg/kg + 7.5 mg/kg(n = 5) Sucralose + Mannitol 144.4 ± 59.9   6.0 ± 1.2*  1.5 mg/kg + 1.5mg/kg (n = 5) Eriodictyol + Mannitol 64.5 ± 35.7*** 3.6 ± 1.1*** 5.0mg/kg + 7.5 mg/kg (n = 4) Eriodictyol + Sucralose 41.1 ± 28.1*** 2.8 ±1.0*** 5.0 mg/kg + 7.5 mg/kg (n = 6) Combinations of three testcompounds Sucralose + Mannitol + Eriodictyol 39.7 ± 21.5*  4.6 ± 0.6***7.5 mg/kg + 7.5 mg/kg + 2.5 mg/kg (n = 6) Data were expressed as means ±SD. Statistical difference resulted from ANOVA and LSD was denoted bywords. *p < 0.05, **p < 0.01, ***p < 0.005, as compared with HFD.Eriodictyol Mannitol Sucralose TG: triglyceride TC: total cholesterol

The results showed that TG and TC increased in liver of mice inducedwith fatty liver. Among the test compounds administered separately,hesperitin, puerarin, eriodictyol, phloridzin, mannitol, menthol, andsucralose could reduce TG in liver significantly. In particular, anexcellent effect of about 67% reduction in liver TG content (p<0.005)was achieved after 4-week treatment of eriodictyol. In addition,hesperitin, eriodictyol, phloridzin, mannitol, menthol, sucralose, andsaccharin could reduce TC in liver significantly. Specifically, anexcellent effect of about 56% reduction in liver TC content (p<0.005)was achieved after 4-week treatment of saccharin.

When the combination of two test compounds was administered, thecombination of saccharin and mannitol, the combination of menthol andmannitol, the combination of sucralose and mannitol, the combination oferiodictyol and mannitol, or the combination of eriodictyol andsucralose could reduce liver TG significantly. In particular, anexcellent effect of about 77% reduction in liver TG content (p<0.005)could be achieved after 4-week treatment of the combination of mentholand mannitol; and an excellent effect of about 78% reduction in liver TGcontent (p<0.005) could be achieved after 8-week treatment of thecombination of eriodictyol and sucralose. In addition, the combinationof sucralose and mannitol, the combination of eriodictyol and mannitol,or the combination of eriodictyol and sucralose could reduce liver TCcontent significantly, in which an excellent effect of about 77%reduction in liver TC content (p<0.005) could be achieved after 8-weektreatment of the combination of eriodictyol and sucralose.

When the combination of three test compounds was administered, thecombination of menthol, mannitol, and eriodictyol or the combination ofsucralose, mannitol, and eriodictyol could reduce liver TGsignificantly. In particular, an excellent effect of about 79% reductionin liver TG content (p<0.005) could be achieved after 8-week treatmentof the combination of sucralose, mannitol, and eriodictyol. In addition,the combination of sucralose, mannitol, and eriodictyol could reduceliver TC significantly.

6.2.2.3 the Test Compounds are Effective in Reducing Liver Damage

6.2.2.3.1 Effects of Reduction in Liver Fat and Liver Damage of LiverTissue

The results of animal experiments showed that a plurality of testcompounds exhibited the efficacies of liver fat and liver tissue damagereduction during the test period of 4 weeks. FIG. 5 showed liver tissuedamage of animals having fatty liver. The liver tissue damage includedmany large vesicular fat droplets covering liver cells near hepaticportal area (including the bile duct, portal vein, hepatic artery) andhepatocellular ballooning. By comparison, after being treated bysilymarin, menthol, eriodictyol, or mannitol for 4 weeks, largevesicular fat droplets within liver cells in liver tissue section weresignificantly reduced. A portion of small broken droplets was stillobserved in mice treated with silymarin, but the liver tissue type ofmice treated with menthol, eriodictyol, or mannitol was close to that ofanimals in Blank group, indicating mild fatty liver diseases.Furthermore, the result of NAS scoring was shown in Table 9.

TABLE 9 The test compounds could reduce the condition of liver damage inanimals NAS Unit Items mg/g liver Blank (n = 13) 0.7 ± 0.5*** HFD (n =12) 3.3 ± 1.7 Positive Control Silymarin 5.0 mg/kg (n = 6) 0.8 ± 0.4***Silymarin 1.5 mg/kg (n = 6) 1.5 ± 0.8* Single test compound Phloridzin2.5 mg/kg (n = 6) 1.8 ± 1.0 Eriodictyol 5.0 mg/kg (n = 6) Eriodictyol2.5 mg/kg (n = 6) 1.5 ± 0.8* Eriodictyol 7.5 mg/kg (n = 6) 1.8 ± 1.1Eriodictyol 1.5 mg/kg (n = 6) 1.8 ± 2.0 Menthol 1.5 mg/kg (n = 6) 1.8 ±1.6 Mannitol 7.5 mg/kg (n = 6) 1.7 ± 0.8* Mannitol 4.5 mg/kg (n = 6) 2.7± 1.9 Mannitol 1.5 mg/kg (n = 6) 1.3 ± 0.8* Saccharin 1.5 mg/kg (n = 3)Puerarin 2.5 mg/kg (n = 6) Hesperitin 2.5 mg/kg (n = 6) 1.7 ± 0.5Combinations of two test compounds Saccharin + Mannitol 1.5 mg/kg + 1.5mg/kg (n = 6) Menthol + Mannitol 2.2 ± 1.5 4.5 mg/kg + 4.5 mg/kg (n = 6)Menthol + Mannitol 0.7 ± 0.5*** 1.5 mg/kg + 1.5 mg/kg (n = 6) Menthol +Mannitol 2.5 ± 1.8 .5 mg/kg + .5 mg/kg (n = 6) Combinations of threetest compounds Menthol + Mannitol + Eriodictyol 2.0 ± 1.4 .5 mg/kg + .5mg/kg + .8 mg/kg (n = 6) Data were expressed as means ± SD. Statisticaldifference resulted from ANOVA and LSD was denoted by words. *p < 0.05,**p < 0.01, ***p < 0.005, as compared with HFD. Hesperitin PuerarinEriodictyol Phloridzin Mannitol Menthol Sucralose Saccharin

NAS (Nonalcoholic Fatty Liver Disease Activity Score) indicated theactivity score of non-alcoholic fatty liver diseases [Hepatology. 2005June; 41(6):1313-21], and comprehensively evaluated the degree ofsteatosis, lobular inflammation, and hepatocyte ballooning. The scoresheet was shown in Table 10. Higher score indicated severer liverdamage.

TABLE 10 NAS Evaluation Project Items Score Degree Definition andDescription Steatosis 0  <5% Refers to amount of surface area involvedby steatosis as evaluated on low to medium power examination; minimalsteatosis (,5%) receives a score of 0 to avoid giving excess weight tobiopsies with very little fatty change 1    5-33% 2 >33-66% 3 >66%Lobular 0 No foci Acidophil bodies are not included in this assessment,inflammation nor is portal inflammation 1 <2 foci/200x 2 2-4 foci/200x3 >4 foci/200x Hepatocyte 0 None ballooning 1 few balloon The term “few”means rare but definite ballooned cells hepatocytes as well as casesthat are diagnostically borderline. 2 Many cells/ Most cases withprominent ballooning also had prominent mallory's hyaline, but Mallory'shyaline is not scored ballooning separately for the NAS.

The results showed that liver tissue damage occurred in mice inducedwith fatty liver (NAS increasing). Among the test compounds administeredseparately, eriodictyol and mannitol could reduce liver damagesignificantly. It is notable that when the combination of two compoundswas administered, the combination of menthol and mannitol achieved anexcellent effect. There was hardly any liver damage appearing. The NASwas the same with that of the Blank.

6.2.2.3.2 Effects of Reduction in Liver Dysfunction

The results of animal experiments showed that a plurality of testcompounds exhibited the efficacies of liver dysfunction reduction inanimals during administration period of 4 or 8 weeks. The results wereshowed in Table 11-1 and Table 11-2.

TABLE 11-1 Test compounds could reduce liver dysfunction in animals(administration period of 4 weeks) ALT AST Unit Items U/L U/L Blank (n =13) 32.6 ± 16.1*** 112.2 ± 53.9*** HFD (n = 12) 70.1 ± 45.2 156.8 ±100.8 Positive Control Silymarin 5.0 mg/kg (n = 6) 33.9 ± 9.3*** 168.1 ±42.6 Silymarin 1.5 mg/kg (n = 6) 43.8 ± 18.7* 153.6 ± 62.5 Single testcompound Mannitol 7.5 mg/kg (n = 6) 25.0 ± 10.8***  63.3 ± 7.7***Mannitol 4.5 mg/kg (n = 6) 44.5 ± 15.9* 107.6 ± 54.3 Mannitol 1.5 mg/kg(n = 6) 40.8 ± 11.4* 187.2 ± 142.1 Sucralose 7.5 mg/kg (n = 6) 32.3 ±10.1**  74.3 ± 18.6** Sucralose 1.5 mg/kg (n = 6) 30.9 ± 16.8*** 127.0 ±31.2 Eriodictyol 5.0 mg/kg (n = 5) 41.4 ± 6.3* 161.4 ± 42.3 Eriodictyol2.5 mg/kg (n = 6) 33.7 ± 18.5*** 100.9 ± 42.0 Puerarin 2.5 mg/kg (n = 6)34.4 ± 14.7***  66.9 ± 8.5*** Phloridzin 2.5 mg/kg (n = 6) 35.7 ± 9.1***161.9 ± 96.2 Hesperitin 2.5 mg/kg (n = 6) 36.8 ± 22.1**  72.4 ± 11.2***Menthol 1.5 mg/kg (n = 6) 41.5 ± 13.7* 129.9 ± 37.1 Saccharin 1.5 mg/kg(n = 3) 50.7 ± 29.7 170.4 ± 28.6 Combinations of two test compoundsMenthol + Mannitol 23.9 ± 17.8***  60.4 ± 8.2*** .5 mg/kg + .5 mg/kg (n= 6) Menthol + Mannitol 16.7 ± 4.3***  59.8 ± 7.5*** 1.5 mg/kg + 1.5mg/kg (n = 6) Sucralose + Mannitol 45.5 ± 15.2  91.4 ± 21.8* 7.5 mg/kg +7.5 mg/kg (n = 6) Sucralose + Mannitol 52.4 ± 34.0  92.1 ± 23.0* 1.5mg/kg + 1.5 mg/kg (n = 6) Eriodictyol + Mannitol 43.4 ± 10.5 151.0 ±54.2 5.0 mg/kg + 7.5 mg/kg (n = 4) Eriodictyol + Sucralose 38.2 ± 10.9*143.8 ± 67.6 5.0 mg/kg + 7.5 mg/kg (n = 4) Saccharin + Mannitol 51.7 ±54.2  70.0 ± 27.6*** 1.5 mg/kg + 1.5 mg/kg (n = 6) Combinations of threetest compounds Menthol + Mannitol + Eriodictyol 21.2 ± 8.7***  54.8 ±13.2*** .5 mg/kg + .5 mg/kg + .8 mg/kg (n = 6) Data were expressed asmeans ± SD. Statistical difference resulted from ANOVA and LSD wasdenoted by words. *p < 0.05, **p < 0.01, ***p < 0.005, as compared withHFD. Hesperitin Puerarin Hesperitin Puerarin Eriodictyol PhloridzinMannitol Menthol Sucralose Saccharin ALT: alanine aminotransferase AST:aspartate transaminase

TABLE 11-2 Test compounds could reduce liver dysfunction in animals(administration period of 8 weeks) ALT AST Unit Items U/L U/L Blank (n =7)   65.1 ± 21.5*** 22.6 ± 4.3***  HFD (n = 8) 111.0 ± 26.2 109.4 ±46.4   Combinations of two test compounds Sucralose + Mannitol  92.4 ±16.5 49.5 ± 14.4*** 7.5 mg/kg + 7.5 mg/kg (n = 5) Sucralose + Mannitol112.5 ± 23.8 93.0 ± 26.0   1.5 mg/kg + 1.5 mg/kg (n = 4) Combinations ofthree test compounds Sucralose + Mannitol + 40.0 ± 12.2*** Eriodictyol7.5 mg/kg + 7.5 mg/kg + 2.5 mg/kg (n = 6) Data were expressed as means ±SD. Statistical difference resulted from ANOVA and LSD was denoted bywords. *p < 0.05, **p < 0.01, ***p < 0.005, as compared with HFD.Mannitol Sucralose ALT: alanine aminotransferase AST: aspartatetransaminase

ALT and AST are most commonly used as enzyme indicators to reflect thebiochemical dysfunction of liver. Under normal circumstances, theseenzymes present in liver cells. However, when liver cells are damaged,they will leak. Increases of serum ALT and AST values generally reflectliver inflammation and liver dysfunction.

The results showed that animals induced with fatty liver (ALT and ASTvalues increasing) suffered from liver dysfunction. Among the testcompounds administered separately, all the hesperitin, puerarin,eriodictyol, phloridzin, mannitol, menthol, sucralose, and saccharincould reduce ALT and AST values significantly. In particular, excellenteffects of about 64% reduction in ALT value (p<0.005) and about 60%reduction in AST value (p<0.005) could be achieved after 4-weektreatment of mannitol.

When the combination of two test compounds was administered, both thecombination of menthol and mannitol, and the combination of eriodictyoland sucralose could reduce ALT value significantly. Also, thecombination of menthol and mannitol, the combination of sucralose andmannitol, or the combination of saccharin and mannitol could reduce ASTvalue significantly. In particular, excellent effects of about 76%reduction in ALT value (p<0.005) and about 62% reduction in AST value(p<0.005) could be achieved after 4-week treatment of the combination ofmenthol and mannitol.

When the combination of three test compounds was administered, thecombination of sucralose, mannitol, and eriodictyol could reduce ALTvalue significantly (p<0.005).

6.2.2.4 the Test Compounds can Improve Liver Antioxidant Activity

The results of animal experiments showed that a plurality of testcompounds exhibited the efficacies of liver antioxidant activityimprovement in animals during the test period of 4 weeks. The resultswere showed in Table 12-1 and Table 12-2.

TABLE 12-1 Test compounds could improve liver antioxidant activity inanimals (Gpx and GSH) Gpx GSH Unit Items U/L U/L Blank (n = 10) 2588.0 ±524.5 1224.1 ± 95.5 HFD (n = 8) 2252.5 ± 395.2 1193.0 ± 203.8 PositiveControl Silymarin 5.0 mg/kg (n = 6) 3358.3 ± 1205.3*** 1398.8 ± 396.5Single test compound Mannitol 7.5 mg/kg (n = 6) 3738.3 ± 665.1*** 2147.7± 459.1*** Mannitol 4.5 mg/kg (n = 6) 3423.3 ± 547.8*** 1605.1 ± 305.9**Mannitol 1.5 mg/kg (n = 6) 2580.0 ± 555.2 1502.5 ± 276.9* Puerarin 2.5mg/kg (n = 6) 3581.7 ± 1056.7*** 1498.1 ± 150.0* Sucralose 7.5 mg/kg (n= 6) 3334.0 ± 377.7** 1609.1 ± 201.1** Sucralose 1.5 mg/kg (n = 6)2995.0 ± 651.1* 1448.0 ± 281.5 Phloridzin 2.5 mg/kg (n = 6) 3234.0 ±505.1** 1387.7 ± 168.2 Hesperitin 2.5 mg/kg (n = 6) 3133.3 ± 376.9*1742.6 ± 241.5*** Eriodictyol 2.5 mg/kg (n = 6) 3083.3 ± 378.9** 1302.0± 241.1 Menthol 1.5 mg/kg (n = 6) 2921.7 ± 640.2 1432.7 ± 104.0 Datawere expressed as means ± SD. Statistical difference resulted from ANOVAand LSD was denoted by words. *p < 0.05, **p < 0.01, ***p < 0.005, ascompared with HFD. Hesperitin Puerarin Hesperitin Puerarin EriodictyolPhloridzin Mannitol Menthol Sucralose Gpx: glutathione peroxidase GSH:glutathione

TABLE 12-2 Test compounds could improve liver antioxidant activity inanimals (Grd and SOD) Grd SOD Unit Items U/L U/L Blank (n = 10) 123.5 ±30.9  380.3 ± 38.8 HFD (n = 8) 82.1 ± 21.7 371.7 ± 49.3 Positive ControlSilymarin 5.0 mg/kg (n = 6) 88.9 ± 29.2  435.9 ± 59.2* Single testcompound Mannitol 7.5 mg/kg (n = 6)  117.6 ± 32.0** 462.8 ± 52.8Mannitol 4.5 mg/kg (n = 6) 110.1 ± 18.4* 429.2 ± 85.2 Mannitol 1.5 mg/kg(n = 6) 95.3 ± 22.1 367.3 ± 35.6 Puerarin 2.5 mg/kg (n = 6) 99.0 ± 17.2434.5 ± 59.8 Sucralose 7.5 mg/kg (n = 6) 90.4 ± 17.2 399.0 ± 34.5Sucralose 1.5 mg/kg (n = 6) 100.0 ± 18.6  373.0 ± 50.4 Phloridzin 2.5mg/kg (n = 6) 82.2 ± 33.6 411.5 ± 87.5 Hesperitin 2.5 mg/kg (n = 6)102.5 ± 28.3  408.3 ± 66.7 Eriodictyol 2.5 mg/kg (n = 6) 86.9 ± 15.7385.9 ± 34.0 Menthol 1.5 mg/kg (n = 6) 95.2 ± 16.2 427.9 ± 41.9 Datawere expressed as means ± SD. Statistical difference resulted from ANOVAand LSD was denoted by words. *p < 0.05, **p < 0.01, ***p < 0.005, ascompared with HFD. Hesperitin Puerarin Hesperitin Puerarin EriodictyolPhloridzin Mannitol Menthol Sucralose Grd: Glutathione reductase SOD:Superoxide dismutase

Gpx, GSH, Grd and SOD are common members of liver antioxidant systemsthat can reduce oxidative stress in the liver and prevent liver fromdamage caused by oxidative stress. Increases of Gpx, GSH, Grd and SODvalues indicate liver maintaining better antioxidant activity.

The results showed that the antioxidant activity of mice induced withfatty liver was reduced. Among the test compounds administeredseparately, all the hesperitin, puerarin, eriodictyol, phloridzin,mannitol, and sucralose could improve antioxidant activitysignificantly. In particular, excellent effects of substantial increasesin Gpx, GSH, Grd, and SOD levels (p<0.005) were achieved after 4-weektreatment of mannitol.

In summary, the compounds as tested including mannitol and sucralose andothers can reduce fat content in the liver, reduce liver damage, andimprove liver antioxidant activity. These compounds had been confirmedsafe through animal experiments and found having potential to bedeveloped into health food or drugs for reducing liver fat andameliorating associated disorders, such as fatty liver diseases, acuteand chronic alcoholic fatty liver diseases, acute and chronicnon-alcoholic fatty liver diseases (NAFLD), acute and chronic alcoholichepatitis, acute and chronic non-alcoholic steatohepatitis,non-alcoholic cirrhosis, and alcoholic cirrhosis (ICD-9-CM diagnosisCodes: 571.8, 571.0, 571.1, 571.2, 571.3, 571.4, 571.5, 571.9).

What is claimed is:
 1. A compound represented by Formula (II):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: (i) R₁ is hydrogen; and R₂ is a C₄-C₁₈ alkyl polyol represented by Formula A: CH₂(CHOH)_(n)CH₂OH  Formula A or a stereoisomer thereof, wherein: n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; or (ii) R₁ is a C₄-C₁₈ alkyl polyol represented by Formula A: CH₂(CHOH)_(n)CH₂OH  Formula A or a stereoisomer thereof, wherein: n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and R₂ is a saccharide group represented by Formula (Ia):

each X is independently —C(═O)—; and m is 3, 4, 5, 6, 7, 8, 9, or
 10. 2. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R₁ is hydrogen; and R₂ is a C₄-C₁₈ alkyl polyol represented by Formula A: CH₂(CHOH)_(n)CH₂OH  Formula A or a stereoisomer thereof, wherein: n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or
 16. 3. The compound of claim 2, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Formula A is:


4. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R₁ is a C₄-C₁₈ alkyl polyol represented by Formula A: CH₂(CHOH)_(n)CH₂OH  Formula A or a stereoisomer thereof, wherein: n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and R₂ is a saccharide group represented by Formula (Ia):


5. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is 2, 3, 4, 5, 6, 7, or
 8. 6. The compound of claim 5, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is
 4. 7. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein m is 3, 4, or
 5. 8. The compound of claim 7, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein m is
 4. 9. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein m is 6, 7, 8, 9, or
 10. 10. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is 9, 10, 11, 12, 13, 14, 15, or
 16. 11. The compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: m is 4; and n is
 4. 12. The compound of claim 1, or a stereoisomer thereof, wherein the compound, or stereoisomer thereof, is selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.
 14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition further comprises one or more additional agents selected from the group consisting of: (i) a first active agent selected from the group consisting of acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, dicalcium phosphate dihydrate, glycerin monostearate, glyceryl behenate, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, lactose monohydrate, lemon oil, magnesium stearate, maltodextrin, mannitol, menthol, methylcellulose, microcrystalline cellulose, oxide red, N-acetylcysteine, pregelatinized starch, saccharin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sodium starch glycolate, sorbic acid, sorbitol, starch acetate, sucralose, Aerosil 200, Brij 35, Brij 58, Brij 76, Copovidone K28, Cremophor EL, Cremophor RH 40, hydrated Dextrates NF, Eudragit S100, Myrj 52, PEG 400, PEG 2000, PEG 4000, PEG 8000, Pluronic F68, Povidone K30, Span 60, Span 80, Tween 20, Tween 40, and Tween 80, or any combination thereof; (ii) a second active agent selected from the group consisting of baicalein, baicalin, butylated hydroxyanisole, (+)-catechin, citric acid, didymin, diosmin, eicosapentaenoic acid, (−)-epicatechin, (−)-epicatechin-3-gallate, (−)-epigallocatechin, ergosterol, eriodictyol, formononetin, galangin, glycerin, glycyrrhizin, gossypin, hesperidin, hesperitin, hyperoside, isovitexin, kaempferol, lemon oil, (+)-limonene, linarin, luteolin, luteolin-7-glucoside, mannitol, menthol, morin, beta-myrcene, myristic acid ethyl ester, naringin, neohesperidin, nordihydroguaiaretic acid, oxide red, phloridzin, poncirin, povidone K-30, pregelatinized starch, protocatechuic acid, puerarin, quercitrin, saccharin, sciadopitysin, silybin, silymarin, sinensetin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sorbic acid, sorbitol, swertiamarin, sucralose, (+)-taxifolin, trans-cinnamic acid, umbelliferone, ursolic acid, wongonin, and xylitol, or any combination thereof; and (iii) any combination of (i) and (ii).
 15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition further comprises one or more additional agents selected from the group consisting of dicalcium phosphate dihydrate, mannitol, menthol, N-acetylcysteine, and sucralose, or any combination thereof.
 16. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition further comprises one or more additional agents selected from the group consisting of: (i) a combination of mannitol and saccharin; (ii) a combination of mannitol and menthol; (iii) a combination of mannitol and sucralose; (iv) a combination of mannitol and eriodictyol; (v) a combination of eriodictyol and sucralose; (vi) a combination of eriodictyol, mannitol, and menthol; and (vii) a combination of eriodictyol, mannitol, and sucralose.
 17. A method for treating an organ injury in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.
 18. The method of claim 17, wherein the organ injury is a kidney injury or a liver injury.
 19. The method of claim 17, wherein the organ injury is caused by carbon tetrachloride, a lipid, or a therapeutic drug.
 20. The method of claim 19, wherein the therapeutic drug is acetaminophen.
 21. The method of claim 17, wherein the compound, or pharmaceutically acceptable salt or stereoisomer thereof, is administered in combination with one or more additional agents selected from the group consisting of: (i) a first active agent selected from the group consisting of acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, dicalcium phosphate dihydrate, glycerin monostearate, glyceryl behenate, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, lactose monohydrate, lemon oil, magnesium stearate, maltodextrin, mannitol, menthol, methylcellulose, microcrystalline cellulose, oxide red, N-acetylcysteine, pregelatinized starch, saccharin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sodium starch glycolate, sorbic acid, sorbitol, starch acetate, sucralose, Aerosil 200, Brij 35, Brij 58, Brij 76, Copovidone K28, Cremophor EL, Cremophor RH 40, hydrated Dextrates NF, Eudragit S100, Myrj 52, PEG 400, PEG 2000, PEG 4000, PEG 8000, Pluronic F68, Povidone K30, Span 60, Span 80, Tween 20, Tween 40, and Tween 80, or any combination thereof; (ii) a second active agent selected from the group consisting of baicalein, baicalin, butylated hydroxyanisole, (+)-catechin, citric acid, didymin, diosmin, eicosapentaenoic acid, (−)-epicatechin, (−)-epicatechin-3-gallate, (−)-epigallocatechin, ergosterol, eriodictyol, formononetin, galangin, glycerin, glycyrrhizin, gossypin, hesperidin, hesperitin, hyperoside, isovitexin, kaempferol, lemon oil, (+)-limonene, linarin, luteolin, luteolin-7-glucoside, mannitol, menthol, morin, beta-myrcene, myristic acid ethyl ester, naringin, neohesperidin, nordihydroguaiaretic acid, oxide red, phloridzin, poncirin, povidone K-30, pregelatinized starch, protocatechuic acid, puerarin, quercitrin, saccharin, sciadopitysin, silybin, silymarin, sinensetin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sorbic acid, sorbitol, swertiamarin, sucralose, (+)-taxifolin, trans-cinnamic acid, umbelliferone, ursolic acid, wongonin, and xylitol, or any combination thereof; and (iii) any combination of (i) and (ii).
 22. A method for ameliorating a liver disease in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof; wherein the liver disease is caused by fatty liver or a disorder associated with fatty liver.
 23. A method for treating fatty liver in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.
 24. The method of claim 23, wherein the compound, or pharmaceutically acceptable salt or stereoisomer thereof, is administered in combination with one or more additional agents selected from the group consisting of: (i) a first active agent selected from the group consisting of acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, dicalcium phosphate dihydrate, glycerin monostearate, glyceryl behenate, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, lactose monohydrate, lemon oil, magnesium stearate, maltodextrin, mannitol, menthol, methylcellulose, microcrystalline cellulose, oxide red, N-acetylcysteine, pregelatinized starch, saccharin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sodium starch glycolate, sorbic acid, sorbitol, starch acetate, sucralose, Aerosil 200, Brij 35, Brij 58, Brij 76, Copovidone K28, Cremophor EL, Cremophor RH 40, hydrated Dextrates NF, Eudragit S100, Myrj 52, PEG 400, PEG 2000, PEG 4000, PEG 8000, Pluronic F68, Povidone K30, Span 60, Span 80, Tween 20, Tween 40, and Tween 80, or any combination thereof; (ii) a second active agent selected from the group consisting of baicalein, baicalin, butylated hydroxyanisole, (+)-catechin, citric acid, didymin, diosmin, eicosapentaenoic acid, (−)-epicatechin, (−)-epicatechin-3-gallate, (−)-epigallocatechin, ergosterol, eriodictyol, formononetin, galangin, glycerin, glycyrrhizin, gossypin, hesperidin, hesperitin, hyperoside, isovitexin, kaempferol, lemon oil, (+)-limonene, linarin, luteolin, luteolin-7-glucoside, mannitol, menthol, morin, beta-myrcene, myristic acid ethyl ester, naringin, neohesperidin, nordihydroguaiaretic acid, oxide red, phloridzin, poncirin, povidone K-30, pregelatinized starch, protocatechuic acid, puerarin, quercitrin, saccharin, sciadopitysin, silybin, silymarin, sinensetin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sorbic acid, sorbitol, swertiamarin, sucralose, (+)-taxifolin, trans-cinnamic acid, umbelliferone, ursolic acid, wongonin, and xylitol, or any combination thereof; and (iii) any combination of (i) and (ii).
 25. A method for treating hepatotoxicity in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.
 26. The method of claim 25, wherein the hepatotoxicity is caused by carbon tetrachloride, a lipid, or a therapeutic drug.
 27. The method of claim 26, wherein the therapeutic drug is acetaminophen.
 28. The method of claim 25, wherein the compound, or pharmaceutically acceptable salt or stereoisomer thereof, is administered in combination with one or more additional agents selected from the group consisting of: (i) a first active agent selected from the group consisting of acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, dicalcium phosphate dihydrate, glycerin monostearate, glyceryl behenate, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, lactose monohydrate, lemon oil, magnesium stearate, maltodextrin, mannitol, menthol, methylcellulose, microcrystalline cellulose, oxide red, N-acetylcysteine, pregelatinized starch, saccharin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sodium starch glycolate, sorbic acid, sorbitol, starch acetate, sucralose, Aerosil 200, Brij 35, Brij 58, Brij 76, Copovidone K28, Cremophor EL, Cremophor RH 40, hydrated Dextrates NF, Eudragit S100, Myrj 52, PEG 400, PEG 2000, PEG 4000, PEG 8000, Pluronic F68, Povidone K30, Span 60, Span 80, Tween 20, Tween 40, and Tween 80, or any combination thereof; (ii) a second active agent selected from the group consisting of baicalein, baicalin, butylated hydroxyanisole, (+)-catechin, citric acid, didymin, diosmin, eicosapentaenoic acid, (−)-epicatechin, (−)-epicatechin-3-gallate, (−)-epigallocatechin, ergosterol, eriodictyol, formononetin, galangin, glycerin, glycyrrhizin, gossypin, hesperidin, hesperitin, hyperoside, isovitexin, kaempferol, lemon oil, (+)-limonene, linarin, luteolin, luteolin-7-glucoside, mannitol, menthol, morin, beta-myrcene, myristic acid ethyl ester, naringin, neohesperidin, nordihydroguaiaretic acid, oxide red, phloridzin, poncirin, povidone K-30, pregelatinized starch, protocatechuic acid, puerarin, quercitrin, saccharin, sciadopitysin, silybin, silymarin, sinensetin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sorbic acid, sorbitol, swertiamarin, sucralose, (+)-taxifolin, trans-cinnamic acid, umbelliferone, ursolic acid, wongonin, and xylitol, or any combination thereof; and (iii) any combination of (i) and (ii).
 29. A method for protecting liver function in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.
 30. A method for reducing free radical levels in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.
 31. The method of claim 30, wherein the subject has a disease or condition characterized by increased free radical levels selected from the group consisting of an alcohol-related disorder, atherosclerosis, cardiovascular disease, cerebrovascular disease, cirrhosis, coronary artery disease, ethanol-induced oxidant stress, hepatitis, hepatic necrosis, hepatoblastoma, hepatocellular carcinoma, inflammation, insulin resistance, isoniazid toxicity, liver damage, a liver disease, a liver histopathology, necrosis, obesity, obesity-induced oxidant stress, poisoning, a renal disease, a renal histopathology, and tuberculosis.
 32. The method of claim 31, wherein the alcohol-related disorder is alcohol abuse, alcohol withdrawal, alcoholic cirrhosis, alcoholic liver disease, or alcoholism.
 33. The method of claim 31, wherein the cirrhosis is liver cirrhosis.
 34. The method of claim 33, wherein the liver cirrhosis is alcoholic liver cirrhosis.
 35. The method of claim 31, wherein the hepatitis is alcoholic hepatitis, chronic hepatitis, drug-induced hepatitis, halothane hepatitis, nonalcoholic steatohepatitis, or toxic hepatitis.
 36. The method of claim 35, wherein the alcoholic hepatitis is acute alcoholic hepatitis.
 37. The method of claim 35, wherein the chronic hepatitis is chronic hepatitis c.
 38. The method of claim 31, wherein the liver damage is ethanol-induced liver injury, liver cell damage, liver fibrosis, or obesity-induced liver injury.
 39. The method of claim 31, wherein the liver disease is chronic liver disease or fatty liver.
 40. The method of claim 39, wherein the fatty liver is alcoholic fatty liver or fatty liver disease.
 41. The method of claim 31, wherein the necrosis is liver necrosis or renal necrosis.
 42. The method of claim 31, wherein the poisoning is heavy metal poisoning.
 43. The method of claim 31, wherein the renal disease is chronic renal disease or renal cell damage.
 44. The method of claim 30, wherein the compound, or pharmaceutically acceptable salt or stereoisomer thereof, is administered in combination with one or more additional agents selected from the group consisting of: (i) a first active agent selected from the group consisting of acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, dicalcium phosphate dihydrate, glycerin monostearate, glyceryl behenate, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, lactose monohydrate, lemon oil, magnesium stearate, maltodextrin, mannitol, menthol, methylcellulose, microcrystalline cellulose, oxide red, N-acetylcysteine, pregelatinized starch, saccharin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sodium starch glycolate, sorbic acid, sorbitol, starch acetate, sucralose, Aerosil 200, Brij 35, Brij 58, Brij 76, Copovidone K28, Cremophor EL, Cremophor RH 40, hydrated Dextrates NF, Eudragit S100, Myrj 52, PEG 400, PEG 2000, PEG 4000, PEG 8000, Pluronic F68, Povidone K30, Span 60, Span 80, Tween 20, Tween 40, and Tween 80, or any combination thereof; (ii) a second active agent selected from the group consisting of baicalein, baicalin, butylated hydroxyanisole, (+)-catechin, citric acid, didymin, diosmin, eicosapentaenoic acid, (−)-epicatechin, (−)-epicatechin-3-gallate, (−)-epigallocatechin, ergosterol, eriodictyol, formononetin, galangin, glycerin, glycyrrhizin, gossypin, hesperidin, hesperitin, hyperoside, isovitexin, kaempferol, lemon oil, (+)-limonene, linarin, luteolin, luteolin-7-glucoside, mannitol, menthol, morin, beta-myrcene, myristic acid ethyl ester, naringin, neohesperidin, nordihydroguaiaretic acid, oxide red, phloridzin, poncirin, povidone K-30, pregelatinized starch, protocatechuic acid, puerarin, quercitrin, saccharin, sciadopitysin, silybin, silymarin, sinensetin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sorbic acid, sorbitol, swertiamarin, sucralose, (+)-taxifolin, trans-cinnamic acid, umbelliferone, ursolic acid, wongonin, and xylitol, or any combination thereof; and (iii) any combination of (i) and (ii).
 45. A method for modulating cytochrome P450 activity in a subject in need thereof, wherein the method comprises administering to the subject a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or stereoisomer thereof.
 46. The method of claim 45, wherein the subject has a disease or condition characterized by increased cytochrome P450 activity selected from the group consisting of an alcohol-related disorder, atherosclerosis, cardiovascular disease, cerebrovascular disease, cirrhosis, coronary artery disease, ethanol-induced oxidant stress, hepatitis, hepatic necrosis, hepatoblastoma, hepatocellular carcinoma, inflammation, insulin resistance, isoniazid toxicity, liver damage, a liver disease, a liver histopathology, necrosis, obesity, obesity-induced oxidant stress, poisoning, a renal disease, a renal histopathology, and tuberculosis.
 47. The method of claim 46, wherein the alcohol-related disorder is alcohol abuse, alcohol withdrawal, alcoholic cirrhosis, alcoholic liver disease, or alcoholism.
 48. The method of claim 46, wherein the cirrhosis is liver cirrhosis.
 49. The method of claim 48, wherein the liver cirrhosis is alcoholic liver cirrhosis.
 50. The method of claim 46, wherein the hepatitis is alcoholic hepatitis, chronic hepatitis, drug-induced hepatitis, halothane hepatitis, nonalcoholic steatohepatitis, or toxic hepatitis.
 51. The method of claim 50, wherein the alcoholic hepatitis is acute alcoholic hepatitis.
 52. The method of claim 50, wherein the chronic hepatitis is chronic hepatitis c.
 53. The method of claim 46, wherein the liver damage is ethanol-induced liver injury, liver cell damage, liver fibrosis, or obesity-induced liver injury.
 54. The method of claim 46, wherein the liver disease is chronic liver disease or fatty liver.
 55. The method of claim 54, wherein the fatty liver is alcoholic fatty liver or fatty liver disease.
 56. The method of claim 46, wherein the necrosis is liver necrosis or renal necrosis.
 57. The method of claim 46, wherein the poisoning is heavy metal poisoning.
 58. The method of claim 46, wherein the renal disease is chronic renal disease or renal cell damage.
 59. The method of claim 45, wherein the compound, or pharmaceutically acceptable salt or stereoisomer thereof, is administered in combination with one or more additional agents selected from the group consisting of: (i) a first active agent selected from the group consisting of acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, dicalcium phosphate dihydrate, glycerin monostearate, glyceryl behenate, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, lactose monohydrate, lemon oil, magnesium stearate, maltodextrin, mannitol, menthol, methylcellulose, microcrystalline cellulose, oxide red, N-acetylcysteine, pregelatinized starch, saccharin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sodium starch glycolate, sorbic acid, sorbitol, starch acetate, sucralose, Aerosil 200, Brij 35, Brij 58, Brij 76, Copovidone K28, Cremophor EL, Cremophor RH 40, hydrated Dextrates NF, Eudragit S100, Myrj 52, PEG 400, PEG 2000, PEG 4000, PEG 8000, Pluronic F68, Povidone K30, Span 60, Span 80, Tween 20, Tween 40, and Tween 80, or any combination thereof; (ii) a second active agent selected from the group consisting of baicalein, baicalin, butylated hydroxyanisole, (+)-catechin, citric acid, didymin, diosmin, eicosapentaenoic acid, (−)-epicatechin, (−)-epicatechin-3-gallate, (−)-epigallocatechin, ergosterol, eriodictyol, formononetin, galangin, glycerin, glycyrrhizin, gossypin, hesperidin, hesperitin, hyperoside, isovitexin, kaempferol, lemon oil, (+)-limonene, linarin, luteolin, luteolin-7-glucoside, mannitol, menthol, morin, beta-myrcene, myristic acid ethyl ester, naringin, neohesperidin, nordihydroguaiaretic acid, oxide red, phloridzin, poncirin, povidone K-30, pregelatinized starch, protocatechuic acid, puerarin, quercitrin, saccharin, sciadopitysin, silybin, silymarin, sinensetin, sodium benzoate, sodium cyclamate, sodium lauryl sulfate, sorbic acid, sorbitol, swertiamarin, sucralose, (+)-taxifolin, trans-cinnamic acid, umbelliferone, ursolic acid, wongonin, and xylitol, or any combination thereof; and (iii) any combination of (i) and (ii).
 60. The method of claim 59, wherein the one or more additional agents are selected from the group consisting of dicalcium phosphate dihydrate, mannitol, menthol, N-acetylcysteine, and sucralose, or any combination thereof.
 61. The method of claim 59, wherein the one or more additional agents are selected from the group consisting of: (i) a combination of mannitol and saccharin; (ii) a combination of mannitol and menthol; (iii) a combination of mannitol and sucralose; (iv) a combination of mannitol and eriodictyol; (v) a combination of eriodictyol and sucralose; (vi) a combination of eriodictyol, mannitol, and menthol; and (vii) a combination of eriodictyol, mannitol, and sucralose.
 62. The method of claim 59, wherein the compound, or pharmaceutically acceptable salt or stereoisomer thereof, and the one or more additional agents are administered simultaneously or sequentially. 